Beta-L-N4-Hydroxycytosine Deoxynucleosides and their use as Pharmaceutical Agents in the Prophylaxis or Therapy of Viral Diseases

ABSTRACT

The invention relates to β-L-N4-hydroxycytosine nucleo-sides, pharmaceutical agents comprising same, and to the use of said β-L-N4-hydroxycytosine nucleosides and pharmaceutical agents in the prophylaxis or therapy of an infection caused by hepatitis B virus (HBV) or human immunodeficiency virus (HIV). The invention also relates to a method for the preparation of said β-L-nucleoside analogs.

The invention relates to novel β-L-N4-hydroxycytosine nucleosides ofgeneral formula I

wherein:R═H, halogen (F, Cl, Br, I), C₁-C₃ alkyl, and

wherein

R₁═H, F; R₂═H, F, OH, N₃; and

R₃═OH, O-acetyl, O-palmitoyl, alkoxycarbonyl, carbamate, phosphonate,monophosphate, bis(S-acyl-2-thioethyl) phosphate, diphosphate ortriphosphate, and their use as pharmaceutical active substances oragents in the prophylaxis and/or treatment of infections caused inparticular by hepatitis B virus (HBV) and human immunodeficiency virus(HIV).

The β-L-N4-hydroxycytosine nucleosides and the acceptable salts orprodrugs thereof can be used alone or in combination with otherβ-L-nucleosides, with 3-deazauridine or with other anti-HBV-effectivecompounds. Fields of use of the invention are medicine and thepharmaceutical industry.

RELATED ART

HBV is the agent that triggers hepatitis B—an infectious disease, thechronic course of which affects about 350 million people worldwide, andparticularly in Southeast Asia, Africa and South America. In a largenumber of cases, hepatitis B virus infections lead to eventual death asa result of liver function failure. Moreover, the chronic course isassociated with a massively increased risk of primary liver carcinomawhich, in China alone, results in about one million new cases of diseaseeach year.

While the precise mechanism through which HBV can induce liver tumorsremains unknown, it must be assumed that tumor induction is closelyassociated with HBV-induced chronic inflammation, developing cirrhosisand regeneration processes of the liver tissue.

The vaccine produced by genetic engineering, which has been availablefor many years, is not suitable for the treatment of hepatitis B virusinfections because it fails to help persons already infected and isunable to stop the chronic course mentioned above.

In recent years, α-interferon produced by genetic engineering, inparticular, has been found useful in the treatment of HBV infections. Itis a cytokin with broad antiviral and immunomodulating activity.However, it is effective in only about 33% of the patients, entailsconsiderable side effects, and cannot be administered on the oral route.

One nucleoside derivative applied with success and approved by the USFood and Drug Administration, as well as in Germany, is lamivudine(β-L-2′,3′-dideoxy-3′-thiacytidine), also known as thiacytidine (3TC),which has been described by Liotta et al. in U.S. Pat. No. 5,539,116. Itis remarkable for its high efficacy both in HbeAg-positive andHbeAg-negative patients and has scarcely any side effects.

Although rapid decline of HBV DNA and normalization of the alaninetransferase activity in serum is found in such treatment, HBV apparentlycannot be completely eliminated from the liver under such therapy, sothat re-onset of a hepatitis B virus infection is possible in many caseseven after completion of a one-year treatment. Attempts are being madeto prevent the above course by extending the treatment to several years,in the hope that HBV could be eliminated completely (Alberti et al., JMed Virol 2002, 67: 458-462).

However, such therapies are associated with an increasing risk ofresistance to lamivudine, which can be as high as 45-55% after thesecond year of treatment (Liaw et al., Gastroenterology 2000, 119:172-180).

The development of additional effective compounds is therefore an urgentnecessity in order to replace the monotherapy by a combination therapywhich not only can be more effective but can also substantially reducethe risk of resistance, as has been found in long-term treatment of HIVinfections (Richman, Nature 2001, 410: 995-1000; Yeni et al., JAMA 2004,292: 251-265).

Lamivudine belongs to a group of so-called β-L-nucleosides. They areenantiomeric compounds of naturally occurring β-D-nucleosides and, far along time, have been regarded as defying enzymatic metabolization andtherefore as inactive in biological systems.

This dogma was relativized for the first time in 1992 by the findings ofSpadari et al. who had discovered that β-L-thymidine, while not beingreacted by cellular thymidine kinase 1, is a substrate of thecorresponding enzyme of herpes simplex virus 1 (Spadari et al., J MedChem 1992, 35: 4214-4220). It has later been found that β-L-nucleosidescan be substrates or inhibitors not only to some viral, but also to somecellular enzymes (Review: Maury, Antiviral Chem Chemother 2000, 11:165-190).

In the following years, a variety of β-L-nucleoside analogs have beensynthesized in pure form, among which—in addition to the above-mentionedlamivudine (3TC; β-L-2′,3′-dideoxy-3′-thiacytidine; Jeong et al., J MedChem 1993, 36: 181-195)-emtricitabine (L-FTC;β-L-2′,3′-dideoxy-5-fluoro-3′-thiacytidine; Furman et al., AntimicrobAgents & Chemother 1992, 36: 2686-2692),β-L-2′-fluoro-5-methylarabino-furanosyluracil (L-FMAU; clevudine; Chu etal., Antimicrob Agents & Chemother 1995, 39: 979-981),β-L-2′,3′-dideoxycytidine and β-L-2′,3′-dideoxy-5-fluorocytidine (L-ddC,L-ddFC; Lin et al., J Med Chem 1994, 37: 798-803),β-L-2′,3′-dideoxy-2′,3′-didehydrocytidine andβ-L-2′,3′-dideoxy-2′,3′-didehydro-5-fluorocytidine (L-d4C and L-d4FC;Lin et al., J Med Chem 1996, 39: 1757-1759), and β-L-thymidine (L-TdR;telbivudine; by Janta Lipinski et al., J Med. Chem. 1998, 41: 2040-2046;Bryant et al., Antimicrob Agents & Chemother 2001, 45: 229-235) havebeen found to be the most effective and promising inhibitors of HBVreplication in vitro and in vivo, which are remarkable for their—in somecases—extremely low cytotoxicity. Among the D-nucleosides, entecavir(BMS 200475), a carbocyclic deoxyguanosine derivative (Innaimo et al.,Antimicrob Agents & Chemother 1997, 41: 1444-1448), should be mentionedin particular, which has proven to be superior to lamivudine in thetreatment of hepatitis B infections in an initial clinical study (Lai etal., Gastroenterology 2002, 123: 1831-1838).

Another promising purine nucleoside of the D series is2′,3′-dideoxy-3′-fluoroguanosine (Matthes et al., Antimicrob Agents &Chemother 1991, 1254-1257; Hafkemeyer et al., Antimicrob Agents &Chemother 1996, 40: 792-794; Löfgren et al., J Viral Hepat 1996, 3:61-65).

Further syntheses of L-nucleosides have been described in Mugnaini etal., Bioorg Med Chem 2003, 11: 357-366; Marquez et al., J Med Chem 1990,33: 978; Lee et al., Nucleosides & Nucleotides 1999, 18: 537-540; Farajet al., Nucleosides & Nucleotides 1997, 16: 1287-1290; Song et al., JMed Chem 2001, 44: 3985-3993; Kotra et al., J Med Chem 1997, 40: 1944;Choi et al., Organic Lett 2002, 4: 305-307; Gumina et al., Curr Top MedChem 2002, 2: 1065-1086; Holy, Tetrahedron Lett. 1971, 189-193; Holy,Collect Czech Chem Commun 1972, 37: 4072-4082; and, in addition, thefollowing patents describe β-L-nucleosides as potential virustaticagents: Gosselin et al., U.S. Pat. No. 6,395,716, Schinazi et al., US2002-0107221 A1; Chu et al., U.S. Pat. No. 5,565,438, U.S. Pat. No.5,567,688, U.S. Pat. No. 5,587,362, WO 92/18517 of the Yale Universityand University of Georgia Research Foundation, Inc.

In addition to β-L-cytosine nucleosides with non-modified cytosine as inβ-L-deoxycytidine (Bryant et al., Antimicrob Agents & Chemother 2001,45: 229-235), β-L-2′,3′-dideoxycytidine (L-ddC; Lin et al., J Med Chem1994, 37: 798-803), β-L-2′,3′-dideoxy-2′,3′-didehydrocytidine (L-d4C;Lin et al., J Med Chem 1996, 39: 1757-1759),β-L-2′-fluoroarabinofuranosylcytosine (L-FAC; Ma et al., J Med Chem1996, 39: 2835-2843), β-L-arabinofuranosylcytosine (L-AraC; Chu et al.,U.S. Pat. No. 5,567,688),β-L-2′,3′-dideoxy-2′,3′-didehydro-2′-fluorocytidine (L-2′FddeC; Lee etal., J Med Chem 1999, 42: 1320-1328), some 5-modified cytosinederivatives have also been synthesized and investigated, especially5-fluorocytosine derivatives which are either more effective thancompounds with non-modified bases, such asβ-L-2′,3′-dideoxy-2′,3′-didehydro-5-fluorocytidine (L-d4FC; Lin et al.,J Med Chem 1996, 39: 1757-1759), equally effective, such asβ-L-2′,3′-dideoxy-5-fluorocytidine (L-ddFC; Lin et al., J Med Chem 1994,37: 798-803) orβ-L-2′,3′-dideoxy-2′,3′-didehydro-2′-fluoro-5-fluorocytidine(L-2′F-ddeFC; Lee et al., J Med Chem 1999, 42: 1320-1328), lesseffective than β-L-2′-deoxy-5-fluorocytidine (L-FdC; Bryant et al.,Antimicrob Agents & Chemother 2001, 45: 229-235), or exhibit no effectwith respect to HBV replication, such asβ-L-2′-fluoroarabinofuranosyl-5-fluorocytosine (L-FAFC; Ma et al., J MedChem 1996, 39: 2835-2843) or β-L-arabinofuranosyl-5-fluorocytosine(L-AraFC; Griffon et al., Eur J Med Chem 2001, 36: 447-460).

Likewise, the following 5-chloro-, bromo- and methyl-modified L-cytosinenucleosides have been described as ineffective or sparingly effective:β-L-deoxy-5-chlorocyticine (CldC; Bryant et al., Antimicrob Agents &Chemother 2001, 45: 229-235),β-L-2′-fluoroarabinofuranosyl-5-chlorocytidine,β-L-2′-fluoroarabinofuranosyl-5-bromocytosine (L-FAC1C, L-FABrC; Ma etal., J Med Chem 1996, 39: 2835-2843),β-L-2′,3′-dideoxy-3′-thia-5-methylcytidine,β-L-2′,3′-dideoxy-3′-thia-5-bromocytidine,β-L-2′,3′-dideoxy-3′-thia-5-chlorocytidine andβ-L-2′,3′-dideoxy-3′-fluoro-5-methylcytidine (Dong et al., Proc NatlAcad Sci USA 1991, 88: 8495-8499; Matthes et al., unpublished) and, inaddition, some β-L-5-methylcytosine nucleosides have been described aseffective to HBV infections (Matthes et al. PCT patent applicationPCT/DE2004/002051).

Some of the above-mentioned L-nucleosides are not only effectiveinhibitors of HBV replication, but also of HIV replication. Thus, forexample, lamivudine has also been approved for the treatment of HIVinfections. Other β-L-cytosine nucleosides already mentioned above, suchas L-ddC, L-d4C, L-d4FC, and FTC, are also strong inhibitors of HIVreplication, whose importance for therapy is to have new effectivecompounds available for combination therapy, thus providing thecapability of coping with development of resistance (Menendez-Arias,Trends Pharmacol Sci 2002, 23: 381-388).

In addition, there are a number of β-L-nucleosides inhibiting HBVreplication only (e.g. L-FMAU, L-TdR, L-CdR, L-3′FddC, L-d4C) and othersinhibiting HIV replication only (e.g. abacavir).

All of the above-mentioned β-L-nucleosides are incorporated by HBV- orHIV-infected cells and must be converted into the nucleosidetriphosphates by cellular enzymes. As a rule, this takes place in astep-by-step fashion. Instead of the nucleosides, however, it is alsopossible to use suitable nucleoside monophosphate triesters wherein thetwo negative phosphate charges are masked by ester bonds, allowingincorporation of said nucleoside monophosphate triesters in cells.Esterases in the cell liberate the nucleoside monophosphate therefrom,so that the first necessary and sometimes absent phosphorylation step ofthe nucleoside is circumvented in the cell in this way. Phosphoricdiesters, e.g. linked with S-acyl-2-thioethyl groups (SATE), were foundto be suitable nucleoside monophosphate prodrugs (Lefebvre et al., J MedChem 1995, 38: 3941-3950; Peyrottes et al., Mini Rev Med Chem 2004, 4:395-408).

It is only in the form of triphosphates where the nucleosides can bindtheir actual target, i.e. the HBV DNA polymerase or reversetranscriptase, in competition with normal substrates and give stronginhibition. As a consequence, the viral genomes can no longer bysynthesized, and virus production comes to a standstill. Such inhibitionmust be selective, i.e., must be restricted to the viral polymerases andmust not co-involve the cellular DNA polymerases, because otherwise—as aconsequence of inhibition of the synthesis of cellular DNA—growth ofrapidly proliferating cells would be impaired.

The invention is based on the object of developing new, antivirallyeffective β-L-N4-hydroxycytosine nucleosides effective against hepatitisB virus infections and HIV infections and exhibiting high efficacyagainst said infections, while having good tolerability and lowtoxicity.

Surprisingly, new β-L-N4-hydroxycytosine deoxynucleoside derivativesaccording to general formula I

wherein:R═H, halogen (F, Cl, Br, I), C₁-C₃ alkyl, and

wherein

R₁H, F; R₂H, F, OH, N₃; and

R₃═OH, O-acetyl, O-palmitoyl, alkoxycarbonyl, carbamate, phosphonate,monophosphate, bis(S-acyl-2-thioethyl) phosphate, diphosphate ortriphosphate,exhibit high antiviral activity against HBV and HIV.

Preferred are β-L-nucleosides in accordance with general formula I,wherein

R═H, F, Cl, Br, I or CH₃, and Z and R₁, R₂ and R₃ have theabove-mentioned meanings.

Particularly preferred are β-L-nucleosides in accordance with generalformula I, wherein

R═H, F or CH₃, and Z has the above-mentioned meanings, andR₁═H or F, preferably H,

R₂═H, F, OH or N₃, and R₃═OH.

The following were found to be particularly effective:

-   β-L-N4-hydroxydeoxycytidine (L-HyCdR),-   β-L-5-methyl-N4-hydroxydeoxycytidine (L-HyMetCdR),-   β-L-5-fluoro-N4-hydroxydeoxycytidine (L-HyFCdR),-   β-L-2′ 3′-dideoxy-N4-hydroxycytidine (L-HyddC),-   β-L-2′, 3′-dideoxy-5-fluoro-N4-hydroxycytidine L-HyddFC)-   β-L-2′,3′-didehydro-2′, 3′-dideoxy-N4-hydroxycytidine (L-HyddeC),-   β-L-2′,3′-didehydro-2′,3′-dideoxy-5-fluoro-N4-hydroxycytidine    (L-HyddeFC),-   β-L-2′ 3′-didehydro-2′ 3′-dideoxy-5-methyl-N4-hydroxycytidine    (L-ddeMetC),-   β-L-2′,3′-didehydro-2′,3′-dideoxy-2′-fluoro-N4-hydroxycytidine    (L-HyFddeC),-   β-L-2′,3′-dideoxy-3′-thia-N4-hydroxycytidine (Hy3TC),-   β-L-2′ 3′-dideoxy-3′-thia-5-fluoro-N4-hydroxycytidine (HyFTC),-   β-L-3′-azido-2′,3′-dideoxy-N4-hydroxycytidine (L-N₃HyCdR),-   β-L-3′-azido-2′,3′-dideoxy-5-fluoro-N4-hydroxycytidine (L-N₃HyFCdR),-   β-L-3′-azido-2′,3′-dideoxy-5-methyl-N4-hydroxycytidine,-   β-L-3′-fluoro-2′,3′-dideoxy-N4-hydroxycytidine (L-FHyCdR).

In the β-D series, N4-hydroxydeoxycytidine has been known for manyyears. However, its rapid cleavage into cytosine and uracil hasprevented in vivo utilization of its effects on cell proliferation(Nelson et al., Mol Pharmacol of 1966, 2: 248-258). Strong inhibition ofthymidylate synthase has been described as cause of theantiproliferative effects (Goldstein et al., J Med Chem 1984, 27:1259-1262), and this has led to the synthesis of other derivatives ofβ-D-N4-hydroxydeoxycytidine, namely, 5-halogen- and5-hydroxymethyl-modified analogs which are also inhibitors ofthymidylate synthase (Rode et al., Biochemistry 1990, 29: 10835-10842;Felczak et al., J Med Chem 2000, 43: 4647-4656).β-D-5-methyl-N4-hydroxydeoxycytidine and, in particular, theribonucleoside β-D-N4-hydroxycytidine haze become known through theirmutagenic effect in bacteria (Janion, Mut Res 1978, 56: 225-234;Sledziewska et al., Mut Res 1980, 70: 11-16).

More recently, said ribonucleoside, i.e., β-D-N4-hydroxycytidine, wasfound to be a strong inhibitor of the replication of hepatitis C virus(HCV) and bovine viral diarrhoea virus (BVDV) (Stuyver et al.,Antimicrob Agents Chemother 2003, 47: 244-254), and this has inducedfurther chemical modifications. Thus, β-D-3′-deoxy-N4-hydroxycytidinehas been prepared and, in addition, the 5 position of the pyrimidinering has been modified by halogen, methyl or 5-trifluoromethyl groups.Moreover, the synthesis of the corresponding enantiomeric 5-modifiedβ-L-3′-deoxy-N4-hydroxycytidine derivatives has been described in thesame paper for the first time, and all of the above derivatives werefound to be ineffective to HVC (Hollecker et al., Antiviral ChemChemother 2004, 14: 33-55).

On the other hand, β-L-N4-hydroxycytosine nucleosides as claimed hereinare as yet unknown.

More specifically, the invention is therefore directed to the newβ-L-N4-hydroxycytosine nucleosides of general formula I, to theirapplication in the production of pharmaceutical agents, topharmaceutical agents including these compounds, and to pharmaceuticalagents including said compounds in combination with otherpharmaceuticals, particularly in combination preparations with3-deazauridine. Simultaneous application e.g. with 3-deazauridinesignificantly increases the efficacy.

3-Deazauridine activates the cellular deoxycytidine kinase and, inaddition, the triphosphate thereof, formed intracellularly, is capableof inhibiting the cellular CTP synthase (Gao et al., NucleosidesNucleotides Nucleic Acids 2000, 19: 371-377). As a consequence of theabove two effects on the cellular deoxycytidine metabolism,3-deazauridine gives rise to increased triphosphate levels of theβ-L-N4-hydroxycytosine nucleosides of the invention, thereby massivelyincreasing their efficacy with respect to HBV and HIV replication.

Surprisingly, it was found that the nucleosides according to theinvention, i.e., the β-L-Hydroxycytosine nucleosides, can be used withhigh antiviral activity against selected viruses, especially againsthepatitis viruses, preferably against hepatitis B virus.

In a preferred embodiment of the invention, derivatives of the inventivenucleosides are used. This may concern structures having modificationswhich, in particular, increase the antiviral activity. However, this mayalso concern a salt, a phosphonate, a monophosphate, a diphosphate, atriphosphate, an ester or a salt of such ester. Advantageously, suchcompounds can be used effectively in antiviral prophylaxis and therapyand exhibit only minor or no side effects at all.

The preparation of the compounds according to the invention is effectedby means of per se known procedures, using modification of β-L-uridineor β-L-thymidine or condensation of modified β-L-sugars with aheterocycle such as 5-fluorouracil (Horwitz et al., J Org Chem 1967, 32:817-818; Martin et al., J Med Chem 1990, 33: 2137-2145; Warshaw et al.,J Med Chem 1990, 33: 1663-1666).

It is possible, for example, that the nucleosides in combination withother therapeutic, preferably antiviral agents have a synergistic effectby increasing the therapeutic effect in an additive or non-additivefashion, particularly by increasing the therapeutic index and/orreducing the risk of toxicity inherent in each single compound.Accordingly, the nucleosides of the invention preferably can also beused in combination therapies, including a wide variety of combinationswith well-known therapeutic agents and pharmaceutically acceptablecarriers. Of course, veterinary uses are also possible, as well as feedadditives for all vertebrates. Particularly preferred is the use inhumans. According to the explications above, the nucleosides of theinvention can be used as drugs in a particularly preferred fashion. Tothis end, the nucleosides can be used alone, as a salt or derivative oras a composition. Pharmaceutically tolerable salts of compounds of thepresent invention include those derived from pharmaceutically tolerableinorganic and organic acids and bases. Examples of suitable acidsinclude hydrochloric, hydrobromic, sulfuric, nitric, perchloric,fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic,p-toluenesulfonic, tartaric, acetic, citric, methanesulfonic,ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic andbenzenesulfonic acids. Preferred acids include hydrochloric, sulfuric,methanesulfonic and ethanesulfonic acids. Most preferred ismethanesulfonic acid. Other acids, such as oxalic acid, although notbeing pharmaceutically tolerable themselves, can be used in theproduction of salts usable as intermediate products in obtaining thecompounds of the invention and their pharmaceutically tolerable acidaddition salts.

Salts derived from suitable bases include alkali metal (e.g. sodium),alkaline earth metal (e.g. magnesium), ammonium and N(C₁₋₄ alkyl)₄ ⁺salts.

Combinations of substituents and variables presented by this inventionare preferably those resulting in the formation of stable compounds. Theterm “stable” as used herein relates to compounds having sufficientstability to allow preparation and maintain the integrity of thecompound for a period of time sufficient to allow the use thereof forthe purposes described in detail herein (for example, therapeutic orprophylactic administration to a mammal or use inaffinity-chromatographic applications). Typically, such compounds arestable for at least one week at a temperature of 40° C. or less and inabsence of moisture or other chemically reactive conditions.

The compounds of the present invention can be used in the form of saltsderived from inorganic or organic acids. For example, such acid saltsinclude the following: acetate, adipate, alginate, aspartate, benzoate,benzenesulfonate, bisulfate, citrate, camphorate, camphersulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate,hexanoate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate,persulfate, 3-phenylpropionate, picrate, pivalate, propionate,succinate, tartrate, thiocyanate, tosylate and undecanoate.

The invention also relates to nucleic acids or oligonucleotidecontaining as building blocks one or more nucleosides of the invention.Such nucleic acids can be produced according to methods well-known tothose skilled in the art, and in a preferred fashion the nucleic acidsof the invention are constituted of from 2 to 5000, preferably from 10to 100 nucleoside building blocks, more preferably from 20 to 40nucleoside building blocks. The nucleic acids or oligonucleotides of theinvention containing central deoxycytidyl-deoxyguanosine (CpG)dinucleotides which were shown to possess immunostimulatory effects. Theinvention includes immunostimulatory effects of nucleic acids oroligonucleotides in which the deoxycytidine of the CpG motif is replacedby β-L-N4-hydroxydeoxycytidine or β-L-N4-hydroxy-5-fluorodeoxycytidineor β-L-N4-hydroxy-5-methyldeoxycytidine. These nucleic acids oroliogonucleotide can be preferably used for the treatment of cancer,HBV- and HIV-infections, asthma and allergic diseases.

The synthetic nucleic acids or antisense nucleic acids according to theinvention can be present in the form of a therapeutic composition orformulation which can be used to stimulate the immunosystem in cancerpatients, to treat human hepatitis-infections asthma or allergicdiseases. They can be used as part of a pharmaceutical composition incombination with a physiologically and/or pharmaceutically tolerablecarrier. The properties of the carrier will depend on the route ofadministration. In addition to synthetic nucleic acid and carrier, sucha composition may include diluents, fillers, salts, buffers,stabilizers, solvents and other well-known materials. The pharmaceuticalcomposition of the invention may also include other active factorsand/or substances enhancing the inhibition of HBV expression.Furthermore, the pharmaceutical composition of the invention may includeother chemotherapeutical agents for the treatment of liver carcinomas.Such additional factors and/or substances can be incorporated in thepharmaceutical composition in order to create a synergistic effecttogether with the synthetic nucleic acids of the invention or reduceside effects of the synthetic nucleic acids according to the invention.On the other hand, the synthetic nucleic acids of the invention can beincorporated in formulations of a particular anti-HBV or anti-cancerfactor and/or substance to reduce the side effects of said anti-HBVfactor and/or substance.

The pharmaceutical composition of the invention can be pre-sent in theform of a liposome wherein the synthetic nucleic acids of the invention,in addition to other pharmaceutically tolerable carriers, are combinedwith amphipathic substances such as lipids, which are present asmicelles in one form of aggregation, insoluble monolayers, liquidcrystals or lamellar layers present in an aqueous solution. Suitablelipids for a liposomal formulation include—but are not limitedto—monoglycerides, diclycerides, sulfatides, lysolecithin,phospholipids, saponins, bile acids and the like. The preparation ofsuch Liposomal formulations proceeds in a per se known manner and iswell-known to those skilled in the art. Furthermore, the pharmaceuticalcomposition of the invention may include other lipid carriers such aslipofectamine or cyclodextrins and the like, thereby enhancing thesupply of said nucleic acids to the cells, or it may include polymerswith delayed release.

The invention also relates to a pharmaceutical agent comprising at leastone nucleoside and/or nucleic acid according to the invention,optionally together with conventional auxiliaries, preferably carriers,adjuvants and/or vehicles. A pharmaceutical agent in the meaning of theinvention is any agent in the field of medicine, which can be used inthe prophylaxis, diagnosis, therapy, follow-up or aftercare of patientswho have come in contact with viruses, including hepatitis viruses, insuch a way that a pathogenic modification of the overall condition or ofthe condition of particular parts of the organism could establish atleast temporarily. Thus, for example, the pharmaceutical agent in themeaning of the invention can be a vaccine, an immunotherapeutic orimmunoprophylactic agent. The pharmaceutical agent in the meaning of theinvention may comprise the nucleosides or nucleic acids of the inventionand/or an acceptable salt or components thereof. For example, salts ofinorganic acids may be concerned, such as phosphoric acid, or salts oforganic acids. Furthermore, the salts can be free of carboxyl groups andderived from inorganic bases, such as sodium, potassium, ammonium,calcium or iron hydroxides, or from organic bases such asisopropylamine, trimethylamine, 2-ethylaminoethanol, histidine andothers. Examples of liquid carriers are sterile aqueous solutionsincluding no additional materials or active ingredients, such as water,or those including a buffer such as sodium phosphate with aphysiological pH value or a physiological salt solution or both, e.g.phosphate-buffered sodium chloride solution. Other liquid carriers maycomprise more than just one buffer salt, e.g. sodium and potassiumchloride, dextrose, propylene glycol, polyethylene glycol or others.

Liquid compositions of said pharmaceutical agents may additionallycomprise a liquid phase, also one excluding water. Examples of suchadditional liquid phases are glycerol, vegetable oils, organic esters orwater-oil emulsions. The pharmaceutical composition or pharmaceuticalagent typically includes a content of at least 0.1 wt.-% of nucleosidesor nucleic acids of the invention, relative to the overallpharmaceutical composition. The respective dose or dose range foradministering the pharmaceutical agent of the invention method is in anamount sufficient to achieve the desired prophylactic or therapeuticantiviral effect. The dose should not be selected in such a way thatindesirable side effects would dominate. In general, the dose will varywith the age, constitution, sex of a patient, and obviously with respectto the severity of the disease. The individual dose can be adjusted bothwith respect to the primary disease and with respect to ensuingadditional complications. The exact dose can be detected by a personskilled in the art, using well-known means and methods, e.g. bydetermining the virus titer as a function of the dose or as a functionof the vaccination scheme or of the pharmaceutical carriers and thelike. Depending on the patient, the dose can be selected individually.For example, a dose of pharmaceutical agent tolerated by a patient canbe one where the local level in plasma or in individual organs rangesfrom 0.1 to 10,000 μM, preferably between 1 and 100 μM. Alternatively,the dose can also be estimated relative to the body weight of thepatient. In this event, for example, a typical dose of pharmaceuticalagent would be adjusted in a range between 0.1 μg to 100 μg per kg bodyweight, preferably between 1 and 50 μg/kg. Furthermore, it is alsopossible to determine the dose with respect to individual organs ratherthan the overall patient. For example, this would apply to those caseswhere the pharmaceutical agent of the invention, incorporated in therespective patient e.g. in a biopolymer, is placed near particularorgans by means of surgery. A number of biopolymers capable ofliberating the nucleosides or nucleic acids in a desired manner arewell-known to those skilled in the art. For example, such a gel mayinclude from 1 to 1000 μg of compounds or pharmaceutical agent of theinvention per ml gel composition, preferably between 5 and 500 μg/ml,and more preferably between 10 and 100 mg/ml. In this event, thetherapeutic agent will be administered in the form of a solid, gel-likeor liquid composition.

In a preferred fashion the pharmaceutical agent may also include one ormore additional agents from the group of antiviral, fungicidal orantibacterial agents and/or immunostimulators. In a preferred fashionthe antiviral agent concerns protease inhibitors and/or reversetranscriptase inhibitors. The immunostimulators are preferablybropirimine, anti-human alpha-interferon antibodies, IL-2, GM-CSF,interferons, diethyl dithiocarbamate, tumor necrosis factors,naltrexone, tuscarasol and/or rEPO.

In another preferred embodiment of the invention the carriers areselected from the group comprising fillers, diluents, binders,humectants, disintegrants, dissolution retarders, absorption enhancers,wetting agents, adsorbents and/or lubricants.

The fillers and diluents are preferably starches, lactose, cane-sugar,glucose, mannitol and silica, the binder is preferablycarboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, thehumectant is preferably glycerol, the disintegrant is preferably agar,calcium carbonate and sodium carbonate, the dissolution retarder ispreferably paraffin, and the absorption enhancer is preferably aquaternary ammonium compound, the wetting agent is preferably cetylalcohol and glycerol monostearate, the adsorbent is preferably kaolinand bentonite, and the lubricant is preferably talc, calcium andmagnesium stearates and solid polyethylene glycols, or mixtures of thematerials mentioned above.

The invention also relates to vectors, cells and/or organisms having anucleoside of the invention, a nucleic acid of the invention and/or apharmaceutical agent of the invention.

The invention also relates to the use of the nucleosides of theinvention, the nucleic acids of the invention and/or the pharmaceuticalagent of the invention in the prophylaxis or therapy of a viral,bacterial, fungicidal and/or parasitic infection or of cancer. Forexample, it is well-known to those skilled in the art that viruses caninduce various tumors. Using the compounds of the invention, such tumorscan be prevented prophylactically or treated therapeutically. Obviously,the structures of the invention can also be utilized in an anticancercombination therapy, for example. Those skilled in the art are alsofamiliar with the fact that, in addition to viruses, bacteria associatedwith viral diseases or appearing by themselves represent a medicalproblem. Numerous bacteria have resistance to the well-knownantibacterial agents. The compounds of the invention can be used in theprophylaxis and treatment of bacterial infections as well. Furthermore,the compounds of the invention can be used in the production of drugsfor the treatment and prophylaxis of bacterial infections. In apreferred fashion the bacteria can be those from the genuses Escherichiacoli, Salmonella spp., Shigella flexneri, Citrobacter freundii,Klebsiella pneumoniae, Vibrio spp., Haemophilus influenzae, Yersiniaenterolitica, Pasturella haemolytica, and Proteus spp.

In another preferred embodiment the invention relates to the use of thecompounds of the invention to prevent incorporation of other nucleosidesduring transcription in a growing DNA chain, prevent formation of aDNA-RNA hybrid, separate a base pair, or in competitive inhibition of agrowing DNA chain.

In another preferred embodiment of the invention, the compounds of theinvention are used in a prophylactic or therapeutic treatment of viraldiseases associated with one of the following viruses or a combinationthereof: hepatitis virus, HIV, bovine immunodeficiency virus, human Tcell leukemia virus, feline immunodeficiency virus, caprinearthritis-encephalitis virus, equine infectious anemia virus, ovineMaedi-Visna virus, Visna-Lenti virus and others. In a preferred fashion,DNA viruses are treated. Those skilled in the art are familiar with thefact that the incidence of such viral infections can be combined withbacterial, fungicidal, parasitic or other infections.

Such use is particularly preferred in those cases where the hepatitisvirus is a hepatitis B or a hepatitis D virus.

In a likewise particularly preferred fashion the pharmaceutical agent ofthe invention comprises inhibitors of HBV DNA polymerase. Obviously, thepharmaceutical agent for treatment, especially of hepatitis B, mayinclude further effective anti-HBV agents, preferably PMEA(adefovir-dipivoxil), famciclovir, penciclovir, diaminopurine-dioxolane(DAPD), clevudine (L-FMAU), entecavir, interferon or thymosin α1 and/orinhibitors of nucleocapsid formation, particularlyheteroarylpyrimidines.

In a likewise preferred fashion the agents are pegylated.

Moreover, it is particularly preferred that the agent includesadditional agents capable of eliminating the function of cellularproteins essential to HBV growth.

In a likewise particularly preferred fashion, the above agent includesagents against viruses resistant to lamivudine or other cytosinenucleosides, such as emtricitabine (L-FTC), L-ddC, L-ddeC, L-dC and/orelvucitabine (L-fD4C). In a preferred fashion the agent can also beemployed against liver carcinoma diseases triggered by chronichepatitis, particularly by HBV.

In a likewise preferred fashion the β-L-nucleosides enhance the effectof other pharmaceutical agents in a non-additive, additive orsynergistic fashion, increase the therapeutic index and/or reduce therisk of toxicity inherent in the respective compounds.

A preferred HIV in the meaning of the invention is HIV-1 with thesubtypes A to J (HIV-1 group M) in accordance with the prior art subtypeclassification and the distantly related HIV-0 (HIV-1 group 0).Preferred main subtypes are 1A, 1B, 1C and 1D. The subtypes 1E, 1G and1H are closely related to HIV-1A and likewise preferred. The preferredHIV-1A and 1C, as well as 1B and 1D show homology with respect to eachother. The likewise preferred HIV-0 is more heterogeneous than HIV-1 inparticular virus isolates. Classification into subtypes is not possible.Also preferred is HIV-2 which can be classified into the subtypes A toE. It has milder pathogenicity compared to HIV-1 and has thereforespread more slowly. The genetic variability results in changes in theexternal coat proteins. The influence on cytotropism, as well as thequestion to what extent this is accompanied by varying transmissionprobabilities have not been clarified sufficiently. Likewise preferredis treatment of double infections with different subtypes (e.g. B andE).

In a preferred embodiment of the invention the nucleosides of theinvention are used in combination with 3-deazauridine. Combined use mayinvolve simultaneous or time-shifted administration. Such combinedadministration can be effected in a combined agent, for example.

For example, the combined agent in the meaning of the invention can besuch in nature that nucleosides of the invention and 3-deazauridine areincluded together in a solution or solid, e.g. in a tablet. In thisevent, the ratio of nucleosides of the invention and 3-deazauridine mayvary freely. A ratio of nucleosides of the invention and 3-deazauridineranging from 1:10,000 to 10,000:1 is preferred. The ratio of nucleosidesof the invention and 3-deazauridine may vary within this range,depending on the desired application. Of course, said at least twocomponents—nucleosides of the invention and 3-deazauridine—can also beincorporated together in a solution or solid in such a way that releasethereof will proceed in a time-shifted fashion. However, the combinedagent in the meaning of the invention may also be constituted of twoseparate solutions or two separate solids, one solution or solidessentially comprising 3-deazauridine and the other solution or solidessentially comprising the nucleosides of the invention. The twosolutions or solids can be associated with a common carrier or withseparate carriers. For example, the two solutions and/or the two solidscan be present in a capsule as common carrier. Such a formulation of thecombined agent of the invention is advantageous in those cases whereadministration of the nucleosides of the invention and 3-deazauridine isto proceed in a time-shifted manner. That is, the organism is initiallycontacted with nucleosides of the invention, e.g. by infusion or oraladministration, to be contacted with the other component of the combinedagent in a time-shifted manner. Of course, it is also possible toprovide the combined agent by means of conventionalpharmaceutical-technical methods and procedures in such a way that theorganism is initially contacted with 3-deazauridine and subsequentlywith the nucleosides of the invention. Hence, the organism is contactedsequentially with the components of the combined agent. The time periodbetween administration of the two components of the combined agent ofthe invention or the initial release of nucleosides of the invention or3-deazauridine depends on the age, sex, overall constitution of thepatient, the disease, or other parameters which can be determined by theattending physician using prior tests, for example.

In a particularly preferred embodiment of the invention the compounds ofthe invention are used as a prodrug, as feed additive and/or as drinkingwater additive, the use as feed additive and/or drinking water additivebeing preferred in veterinary medicine.

In a particularly preferred fashion the compounds of the invention areused as prodrug. The utilization of endocytosis for the cellular uptakeof active substances comprising polar compounds is highly effective forsome, particularly long-lived substances, but is very difficult totransfer to more general uses. One alternative is the prodrug conceptgenerally known to those skilled in the art. By definition, a prodrugincludes its active substance in the form of a non-active precursormetabolite. It is possible to distinguish between carrier prodrugsystems, some of them being multi-component ones, and biotransformationsystems. The latter include the active substance in a form requiringchemical or biological metabolization. Such prodrug systems arewell-known to those skilled in the art, e.g. valacyclovir as a precursorof acyclovir, or others. Carrier prodrug systems include the activesubstance as such, bound to a masking group which can be cleaved off bya preferably simple controllable mechanism. The inventive function ofmasking groups in the nucleosides of the invention is neutralization ofthe negative charge on the phosphate residue for improved reception bycells. When using the nucleosides of the invention together with amasking group, the latter may also influence other pharmacologicalparameters, such as oral bioavailability, distribution in tissue,pharmacokinetics, as well as stability to non-specific phosphatases. Inaddition, delayed release of the active substance may entail a depoteffect. Furthermore, modified metabolization may occur, therebyachieving higher efficiency of the active substance or organspecificity. In the event of a prodrug formulation, the masking group,or a linker group binding the masking group to the active substance, isselected in such a way that the nucleoside prodrug has sufficienthydrophilicity to be dissolved in the blood serum, sufficient chemicaland enzymatic stability to reach the site of action, and hydrophilicitysuitable for diffusion-controlled membrane transport. Furthermore, itshould permit chemical or enzymatic liberation of the active sub-stancewithin a reasonable period of time and, of course, the liberatedauxiliary components should not be toxic. In the meaning of theinvention, however, the nucleoside with no mask or no linker and no maskcan also be understood as prodrug because the structure inhibiting viralDNA polymerase is a high-energy triphosphate which initially must beprovided via enzymatic and biochemical processes from the incorporatednucleoside in the cell.

In another particularly preferred embodiment of the invention thecompounds of the invention are formulated as a gel, powder, tablet,sustained-release tablet, premix, emulsion, brew-up formulation, drops,concentrate, granulate, syrup, pellet, bolus, capsule, aerosoa, sprayand/or inhalant and/or used in this form. The tablets, coated tablets,capsules, pills and granulates can be provided with conventionalcoatings and envelopes optionally including opacification agents, andcan be composed such that release of the active substance(s) takes placeonly or preferably in a particular area of the intestinal tract,optionally in a delayed fashion, to which end polymer substances andwaxes can be used as embedding materials.

Preferably, the drugs of the present invention can be used in oraladministration in any orally tolerable dosage form, including capsules,tablets and aqueous suspensions and solutions, without being restrictedthereto. In case of tablets for oral application, carriers frequentlyused include lactose and corn starch. Typically, lubricants such asmagnesium stearate can be added. For oral administration in the form ofcapsules, diluents that can be used include lactose and dried cornstarch. In oral administration of aqueous suspensions the activesubstance is combined with emulsifiers and suspending agents. Also,particular sweeteners and/or flavors and/or coloring agents can beadded, if desired.

The active substance(s) can also be present in micro-encapsulated form,optionally with one or more of the above-specified carrier materials.

In addition to the active substance(s), suppositories may includeconventional water-soluble or water-insoluble carriers such aspolyethylene glycols, fats, e.g. cocoa fat and higher esters (forexample, C₁₄ alcohols with C₁₆ fatty acids) or mixtures of thesesubstances.

In addition to the active substance(s), ointments, pastes, creams andgels may include conventional carriers such as animal and vegetablefats, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silica, talc and zinc oxideor mixtures of these substances.

In addition to the active substance(s), powders and sprays may includeconventional carriers such as lactose, talc, silica, aluminum hydroxide,calcium silicate and polyamide powder or mixtures of these substances.In addition, sprays may include conventional propellants such aschlorofluorohydrocarbons.

In addition to the active substance(s), solutions and emulsions mayinclude conventional carriers such as solvents, solubilizers, andemulsifiers such as water, ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils, especially cottonseed oil, peanut oil, corn oil, olive oil, castor oil and sesame oil,glycerol, glycerol formal, tetrahydrofurfuryl alcohol, polyethyleneglycols, and fatty esters of sorbitan, or mixtures of these substances.For parenteral application, the solutions and emulsions may also bepresent in a sterile and blood-isotonic form.

In addition to the active substance(s), suspensions may includeconventional carriers such as liquid diluents, e.g. water, ethylalcohol, propylene glycol, suspending agents, e.g. ethoxylatedisostearyl alcohols, polyoxyethylenes orbitol and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, andtragacanth, or mixtures of these substances.

The drugs can be present in the form of a sterile injectableformulation, e.g. as a sterile injectable aqueous or oily suspension.Such a suspension can also be formulated by means of methods known inthe art, using suitable dispersing or wetting agents (such as Tween 80)and suspending agents. The sterile injectable formulation can also be asterile injectable solution or suspension in a non-toxic, parenterallytolerable diluent or solvent, e-g. a solution in 1,3-butanediol.Tolerable vehicles and solvents that can be used include mannitol,water, Ringer's solution, and isotonic sodium chloride solution.Furthermore, sterile, non-volatile oils are conventionally used assolvents or suspending medium. Any mild non-volatile oil, includingsynthetic mono- or diglycerides, can be used for this purpose. Fattyacids such as oleic acid and glyceride derivatives thereof can be usedin the production of injection agents, e.g. natural pharmaceuticallytolerable oils such as olive oil or castor oil, especially in theirpolyoxyethylated forms. Such oil solutions or suspensions may alsoinclude a long-chain alcohol, such as Ph.Helv., or a similar alcohol asdiluent or dispersant.

The above-mentioned formulation forms may also include colorants,preservatives, as well as odor- and taste-improving additives, e.g.peppermint oil and eucalyptus oil, and sweeteners, e.g. saccharine.Preferably, the active sub-stances of formula (I) and (II), i.e., thenucleosides of the invention, should be present in the above-mentionedpharmaceutical preparations at a concentration of about 0.1 to 99.5wt.-%, more preferably about 0.5 to 95 wt.-% of the overall mixture.

In addition to the compounds of formula (I) and (II), theabove-mentioned pharmaceutical preparations may include furtherpharmaceutical active substances. The production of the pharmaceuticalpreparations specified above proceeds in a usual manner according towell-known methods, e.g. by mixing the active substance(s) with thecarrier material(s).

The above-mentioned preparations can be applied in humans and animals onan oral, rectal, parenteral (intravenous, intramuscular, subcutaneous),intracisternal, intravaginal, intraperitoneal route, locally (powders,ointment, drops) and used in the therapy of infections in hollow areasand body cavities. Injection solutions, solutions and suspensions fororal therapy, gels, brew-up formulations, emulsions, ointments or dropsare possible as suitable preparations. For local therapy, ophthalmic anddermatological formulations, silver and other salts, ear drops, eyeointments, powders or solutions can be used. With animals, ingestion canbe effected via feed or drinking water in suitable formulations.Furthermore, gels, powders, tablets, sustained-release tablets,premixes, concentrates, granulates, pellets, boli, capsules, aerosols,sprays, inhalants can be used in humans and animals. Moreover, thecompounds of the invention can be incorporated in other carriermaterials such as plastics (plastic chains for local therapy) collagenor bone cement.

In another preferred embodiment of the invention the compounds of theinvention, i.e., the nucleosides of the invention, the nucleic acids ofthe invention, the inventive pharmaceutical agents or vectors, cells andorganisms, are incorporated in a preparation at a concentration of 0.1to 99.5, preferably 0.5 to 95, and more preferably 20 to 80 wt.-%. Thatis, the compounds of the invention are pre-sent in the above-specifiedpharmaceutical formulations, e.g. tablets, pills, granulates and others,at a concentration of preferably 0.1 to 99.5 wt.-% of the overallmixture. Those skilled in the art will be aware of the fact that theamount of active substance, i.e., the amount of an inventive compoundcombined with the carrier materials to produce a single dosage form,will vary depending on the host to be treated and on the particular typeof administration. Once the condition of a host or patient has improved,the proportion of active compound in the preparation can be modified soas to obtain a maintenance dose. Depending on the symptoms, the dose orfrequency of administration or both can subsequently be reduced to alevel where the improved condition is retained. Once the symptoms havebeen alleviated to the desired level, the treatment should beterminated. However, patients may require an intermittent treatment on along-term basis if any symptoms of the disease should recur.Accordingly, the proportion of the compounds, i.e. their concentration,in the overall mixture of the pharmaceutical preparation, as well as thecomposition or combination thereof, is variable and can be modified andadapted by a person of specialized knowledge in the art.

Those skilled in the art will be aware of the fact that the compounds ofthe invention can be contacted with an organism, preferably a human oran animal, on various routes. Furthermore, a person skilled in the artwill also be familiar with the fact that the pharmaceutical agents inparticular can be applied at varying dosages. Application should beeffected in such a way that a viral disease is combatted as effectivelyas possible or the onset of such a disease is prevented by aprophylactic administration. Concentration and type of application canbe determined by a person skilled in the art using routine tests.Preferred applications of the compounds of the invention are oralapplication in the form of powders, tablets, juice, drops, capsules orthe like, rectal application in the form of suppositories, solutions andthe like, parenteral application in the form of injections, infusionsand solutions, inhalation of vapors, aerosols and dusts and pads, andlocal application in the form of ointments, pads, dressings, ravages andthe like. Contacting with the compounds according to the invention ispreferably effected in a prophylactic or therapeutic fashion. Inprophylactic administration, an infection with the above-mentionedviruses is to be pre-vented at least in such a way that, followinginvasion of single viruses, e.g. into a wound, further growth thereof ismassively reduced or viruses having invaded are destroyed virtuallycompletely. In therapeutic contacting, a manifest infection of thepatient is already existing, and the viruses already present in the bodyare either to be destroyed or inhibited in their growth. Other forms ofapplication preferred for this purpose are e.g. subcutaneous,sublingual, intravenous, intramuscular, intraperitoneal and/or topicalones.

For example, the suitability of the selected form of application, of thedose, application regimen, selection of adjuvant and the like can bedetermined by taking serum aliquots from the patient, i.e., human oranimal, and testing for the presence of viruses, i.e., determining thevirus titer, in the course of the treatment procedure. Alternatively orconcomitantly, the condition of the liver, but also, the amount of Tcells or other cells of the immune system can be determined in aconventional manner so as to obtain a general survey on theimmunological constitution of the patient and, in particular, theconstitution of organs important to the metabolism, particularly of theliver. Additionally, the clinical condition of the patient can beobserved for the desired effect, especially the anti-infectious,preferably antiviral effect. As set forth above, especially hepatitis,but also HIV or other diseases can be associated with other e.g.bacterial or fungicidal infections or tumor diseases, for which reasonadditional clinical co-monitoring of the course of such concomitantinfections or tumor diseases is also possible. Where insufficienttherapeutic effectiveness is achieved, the patient can be subjected tofurther treatment using the agents of the invention, optionally modifiedwith other well-known medicaments expected to bring about an improvementof the overall constitution. Obviously, it is also possible to modifythe carriers or vehicles of the pharmaceutical agent or to vary theroute of administration. In addition to oral ingestion, e.g.intramuscular or subcutaneous injections or injections into the bloodvessels can be envisaged as another preferred route of therapeuticadministration of the compounds according to the invention. At the sametime, supply via catheters or surgical tubes can also be used. Inaddition to the above-specified concentrations during use of thecompounds of the invention, the compounds in a preferred embodiment canbe employed in a total amount of 0.05 to 500 mg/kg body weight per 24hours, preferably 5 to 100 mg/kg body weight. Advantageously, this is atherapeutical quantity which is used to prevent or improve the symptomsof a disorder or of a responsive, pathologically physiologicalcondition. The amount administered is sufficient to prevent or inhibitinfection or spreading of an infectious agent such as hepatitis B or HIVin the recipient. The effect of the compounds of the invention on theabove-mentioned viruses, with respect to their prophylactic ortherapeutic potential, is seen e.g. as an inhibition of the viralinfection, inhibition of syncytium formation, inhibition of fusionbetween virus and target membrane, as a reduction or stabilization ofthe viral growth rate in an organism, or in another way. For example,the therapeutic effect can be such that, as a desirable side effect,particular antiviral medicaments are improved in their effect or, byreducing the dose, the number of side effects of these medicaments willbe reduced as a result of applying the compounds of the invention. Ofcourse, the therapeutic effect also encompasses direct action on theviruses in a host. That is, however, the effect of the compounds of theinvention is not restricted to eliminating the viruses, but rathercomprises the entire spectrum of advantageous effects in prophylaxis andtherapy. Obviously, the dose will depend on the age, health and weightof the recipient, degree of the disease, type of required simultaneoustreatment, frequency of the treatment and type of the desired effectsand side-effects. The daily dose of 0.05 to 500 mg/kg body weight can beapplied as a single dose or multiple doses in order to furnish thedesired results. The dose levels per day can be used in prevention andtreatment of a viral infection, including hepatitis B infection. Inparticular, pharmaceutical agents are typically used in about 1 to 7administrations per day, or alternatively or additionally as acontinuous infusion. Such administrations can be applied as a chronic oracute therapy. Of course, the amounts of active substance that arecombined with the carrier materials to produce a single dosage form mayvary depending on the host to be treated and on the particular type ofadministration. In a preferred fashion, the daily dose is distributedover 2 to 5 applications, with 1 to 2 tablets including an activesubstance content of 0.05 to 500 mg/kg body weight being administered ineach application. Of course, it is also possible to select a highercontent of active substance, e.g. up to a concentration of 5000 mg/kg.The tablets can also be sustained-release tablets, in which case thenumber of applications per day is reduced to 1 to 3. The activesubstance content of sustained-release tablets can be from 3 to 3000 mg.If the active substance—as set forth above—is administered by injection,the host is preferably contacted 1 to 8 times per day with the compoundsof the invention or by using continuous infusion, in which casequantities of from 1 to 4000 mg per day are preferred. The preferredtotal amounts per day were found advantageous both in human andveterinary medicine. It may become necessary to deviate from theabove-mentioned dosages, and this depends on the nature and body weightof the host to be treated, the type and severity of the disease, thetype of formulation and application of the drug, and on the time periodor interval during which the administration takes place. Thus, it may bepreferred in some cases to contact the organism with less than theamounts mentioned above, while in other cases the amount of activesubstance specified above has to be surpassed. A person of specializedknowledge in the art can determine the optimum dosages required in eachcase and the type of application of the active substances.

In another particularly preferred embodiment of the invention thecompounds of the invention, i.e., the nucleoside, the nucleic acid, thepharmaceutical agent, the vector, the cells and/or organism, are used ina single administration of from 1 to 80, especially from 3 to 30 mg/kgbody weight. In the same way as the total amount per day, the amount ofa single dose per application can be varied by a person of specializedknowledge in the art. Similarly, the compounds used according to theinvention can be employed in veterinary medicine with theabove-mentioned single concentrations and formulations together with thefeed or feed formulations or drinking water. A single dose preferablyincludes that amount of active substance which is administered in oneapplication and which normally corresponds to one whole, one half dailydose or one third or one quarter of a daily dose. Accordingly, thedosage units may preferably include 1, 2, 3 or 4 or more single doses or0.5, 0.3 or 0.25 single doses. In a preferred fashion, the daily dose ofthe compounds according to the invention is distributed over 2 to 10applications, preferably 2 to 7, and more preferably 3 to 5applications. Of course, continuous infusion of the agents according tothe invention is also possible.

In a particularly preferred embodiment of the invention, 1 to 2 tabletsare administered in each oral application of the compounds of theinvention. The tablets according to the invention can be provided withcoatings and envelopes well-known to those skilled in the art or can becomposed in a way so as to release the active substance (s) only inpreferred, particular regions of the host.

In another preferred embodiment of the invention the compounds accordingto the invention can be employed together with at least one otherwell-known pharmaceutical agent. That is to say, the compounds of theinvention can be used in a prophylactic or therapeutic combination inconnection with well-known drugs. Such combinations can be administeredtogether, e.g. in an integrated pharmaceutical formulation, orseparately, e.g. in the form of a combination of tablets, injection orother medications administered simultaneously or at different times,with the aim of achieving the desired prophylactic or therapeuticeffect. These well-known agents can be agents which enhance the effectof the nucleosides according to the invention. In the antibacterialsector, in particular, it was found that a wide variety of antibioticsimprove the effect of nucleosides. This includes agents such asbenzylpyrimidines, pyrimidines, sulfoamides, rifampicin, tobramycin,fusidinic acid, clindamycin, chloramphenicol and erythromycin.Accordingly, another embodiment of the invention relates to acombination wherein the second agent is least one of the above-mentionedantiviral or antibacterial agents or classes of agents. It should alsobe noted that the compounds of tile invention and combinations can alsobe used in connection with immune-modulating treatments and therapies.

Typically, there is an optimum ratio of compound(s) of the inventionwith respect to each other and/or with respect to other therapeutic oreffect-enhancing agents (such as transport inhibitors, metabolicinhibitors, inhibitors of renal excretion or glucuronidation, such asprobenecid, acetaminophen, aspirin, lorazepan, cimetidine, ranitidine,colifibrate, indomethacin, ketoprofen, naproxen etc.) where the activesubstances are present at an optimum ratio. Optimum ratio is defined asthe ratio of compound(s) of the invention to other therapeutic agent(s)where the overall therapeutic effect is greater than the sum of theeffects of the individual therapeutic agents. In general, the optimumratio is found when the agents are present at a ratio of from 10:1 to1:10, from 20:1 to 1:20, from 100:1 to 1:100 and from 500:1 to 1:500. Insome cases, an exceedingly small amount of a therapeutic agent will besufficient to increase the effect of one or more other agents. Inaddition, the use of the compounds of the invention in combinations isparticularly beneficial in order to reduce the risk of developingresistance. Of course, the compounds of the invention, such asnucleosides or nucleic acids, can be used in combination with otherwell-known antiviral agents. Such agents are well-known to those skilledin the art. Accordingly, the compounds of the invention can beadministered together with all conventional agents, especially otherdrugs, available for use particularly in connection with hepatitisdrugs, either as a single drug or in a combination of drugs. They can beadministered alone or in combination with same.

In a preferred fashion the compounds of the invention are administeredtogether with said other well-known pharmaceutical agents at a ratio ofabout 0.005 to 1. Preferably, the compounds of the invention areadministered particularly together with virus-inhibiting agents at aratio of from 0.05 to about 0.5 parts to about 1 part of said knownagents. In this event, tumor-inhibiting or antibacterial agents can beconcerned. The pharmaceutical composition can be present in substance oras an aqueous solution together with other materials such aspreservatives, buffer substances, agents to adjust the osmolarity of thesolution, and so forth.

The invention also relates to the use of the nucleic acids of theinvention as antisense nucleic acids, particularly in an antiviraltherapy. Those skilled in the art are familiar with the fact thatnucleic acids can be used as anti-sense nucleic acids. In a preferredfashion the nucleic acid of the invention serves to preventhybridization of the RNA during translation, and this proceeds viahybridization of the viral RNA with the nucleic acids according to theinvention. More specifically, the nucleic acids of the invention can beused as agents against hepatitis B because degradation thereof bycellular restriction enzymes is absent or difficult. In general, thenucleic acid of the invention hybridizes with the DNA of the hepatitis Bvirus, thereby not only impeding translation, but also transcriptioninto viral DNA.

The nucleosides and nucleic acids according to the invention can be usedin the production of pharmaceutical agents. Thus, the teaching of theinvention may also relate to a method for the treatment of a viral,bacterial, fungicidal and/or parasitic infection or of cancer, in whichmethod the nucleosides and/or nucleic acids of the invention arecontacted with an organism. Treatment in the meaning of the inventionincludes both prophylactic and therapeutic treatment. In a preferredfashion the compounds of the invention can be used to protect organisms,especially human patients, from viral infection during a particularincident, such as delivery, or for a prolonged period of time, in acountry where high risk of hepatitis B infection exists. In such cases,the compounds of the invention can be used alone or together with otherprophylactic agents or other antiviral agents enhancing the efficacy ofthe respective agent. Preferably following oral application, thenucleosides of the invention advantageously can undergo easy absorptioninto the bloodstream of mammals, especially human mammals.Advantageously, the compounds exhibit good water solubility andconsistent oral availability. In particular, it is said good oralavailability that makes the compounds of the invention excellent agentsfor orally administered cures of treatment and prevention against viralinfection, especially hepatitis B infection. Of course, the compounds ofthe invention not only are orally bioavailable, but advantageously havealso a high therapeutic index which, in particular, is a measure oftoxicity versus anti-viral effect. Accordingly, the compounds of theinvention are more effective at lower dose levels compared to selectedwell-known antiviral agents, avoiding the toxic effect associated withthese medical substances. The potential of the compounds of theinvention of being released at doses far exceeding their activeantiviral range is particularly advantageous in slowing down orpreventing possible development of resistant variants. During aprophylactic treatment, in particular, the compounds of the inventioncan be used in a healthy, but also in a virally infected, especially ina hepatitis B virus infected patient, either as a single agent ortogether with other antiviral agents preferably impairing thereplication cycle of hepatitis viruses. The use of the compounds of theinvention in prophylaxis and therapy proceeds in a way well-known tothose skilled in the art. In those cases where the method of treating aviral infection with the nucleosides of the invention represents acombination therapy, each agent used, i.e., both the well-knowncompounds and the compounds of the invention, has an additive,non-additive or synergistic effect in inhibiting virus replication,because action of each agent at a different site of replication of theviruses advantageously can be envisaged. Advantageously, the method ofsuch combination therapies can also reduce the dosage of a conventionalantiviral agent which, in comparison (when administering the agentalone), would be required for a desired therapeutic or prophylacticeffect. Such combinations in the method of the invention for thetreatment of viral diseases can reduce or eliminate the side effects ofconventional therapies using single antiviral agents, and suchcombinations advantageously do not impair but rather synergisticallyincrease the antiviral effect of these agents. These combinations reducethe potential of resistance to therapy using single agents, whileadvantageously minimizing the toxicity associated therewith. Thesecombinations can also increase the efficacy of conventional agentswithout increasing the toxicity associated therewith. In a particularlypreferred fashion the compound according to this invention, togetherwith other antiviral or antibacterial or fungicidal agents, preventreplication of the genetic material of viruses in an additive orsynergistic manner. Inter alia, preferred combination therapies includethe administration of a compound of the invention together with ddC,d4T, 3TC or a combination thereof. Of course, administration togetherwith other nucleoside derivatives or viral reverse transcriptaseinhibitors or protease inhibitors may also be preferred in the method ofthe invention or in the use according to the invention. Jointadministration of the compounds of the invention and viral reversetranscriptase inhibitors or aspartyl protease inhibitors shows anadditive or synergistic effect, thereby preventing, essentially reducingor completely eliminating virus replication or infection or both, orsymptoms associated therewith. Administration of a combination of agentscan be preferred over administration of single agents. The compounds ofthe invention can also be used together with immunomodulators orimmunostimulators; preferred immunomodulators or immunostimulators are:bropirimine, anti-human α-interferon antibodies, IL-2, GK-CSF,interferon α, diethyl dithiocarbamate, tumor necrosis factor,naltrexone, tuscarasol, rEPO and antibiotics such as pentamidineisethioriate, but also agents preventing or combatting malignant tumorsassociated with viral diseases. In the method for the treatment ofviral, bacterial, fungicidal and/or parasitic infections or of cancer,the compounds of the invention—as set forth above—can be administeredtogether with tolerable carriers, adjuvants or vehicles.Pharmaceutically tolerable carriers, adjuvants and vehicles that can beused in the drugs of this invention include ion exchangers, aluminumoxide, aluminum stearate, lecithin, self-emulsifying drug deliverysystems (SEDDS), such as d-α-tocopherol-polyethylene glycol 1000succinate, or other similar polymer delivery matrices, serum proteinssuch as human serum albumin, buffer substances such as phosphates,glycine, sorbic acids, potassium sorbate, partial glyceride mixtures ofsaturated vegetable fatty acids, water, salts or electrolytes such asprotamin sulfate, disodium hydrogen phosphate, potassium hydrogenphosphate, sodium chloride, zinc salts, colloidal silicon dioxide,magnesium trisilicates, polyvinylpyrrolidone, materials on cellulosebasis, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene block polymers,polyethylene glycol, and wool fat, but are not restricted thereto.Cyclodextrins such as α-, β-, and γ-cyclodextrin or chemically modifiedderivatives such as hydroxyalkylcyclodextrins, including 2- and3-hydroxypropyl-β-cyclodextrins or other solubilized derivatives, canalso be used with advantage in order to enhance delivery of thecompounds according to the invention. In the context with this method,the compounds of the invention can be administered orally, parenterally,via inhalation spray, topically, rectally, nasally, buccally, vaginally,or via implanted reservoirs. Oral administration or administration viainjection is a preferred form of contacting. The drugs of this inventionmay include any conventional non-toxic, pharmaceutically tolerablecarriers, adjuvants or vehicles. In some cases, the pH value of theformulation can be adjusted using pharmaceutically tolerable acids,bases or buffers in order to increase the stability of the formulatedcompound or delivery form thereof. The term parenteral, as used herein,includes subcutaneous, intracutaneous, intravenous, intramuscular,intra-articular, intrasynovial, intrasternal, intrathecal, intralesionaland intracranial injection or infusion procedures as a form ofcontacting.

The invention also relates to a kit comprising the compounds of theinvention, optionally together with information on how to combine thecontents of the kit. The information for combining the contents of thekit relates to the use of said kit in the prophylaxis and/or therapy ofdiseases, particularly viral diseases. For example, the information mayalso concern a therapeutic scheme, i.e., a concrete injection orapplication scheme, the dose to be administered, or other.

The nucleoside analogs of the invention have many advantages. In thecourse of their individual development, human and animal organisms mustcope with numerous pathogens. For example, these pathogens can be fungi,bacteria, but also viruses, in particular. Each year, millions of peopleand economically useful animals develop a viral disease, and a largenumber of such infections are accompanied by significant healthimpairments. Untreated for a prolonged period of time, diseases withhuman immunodeficiency virus and hepatitis viruses can be fatal.

The viruses an organism has to cope with strongly differ in theirinfectious potential. Highly infectious viruses include hepatitis Bvirus (HBV) which may cause inflammations of the liver, regularlyaccompanied by liver cell damage, and such liver damage can develop upto a liver tumor in chronic courses with selected viruses, such ashepatitis viruses B, C and D.

To allow successful combatting of viruses in a host organism, e.g. in ahuman or in a farm or domestic animal, the prior art has developedvarious antiviral therapies. A large number of these therapies arechemotherapies intended to prevent replication of pathogenic viruses ina host cell. Various phases of replication, such as adsorption,penetration, translation, transcription of the viral genes, replicationof nucleic acids, as well as assembly of virus particles, are possibleas targets of attack for the so-called virustatic agents used to thisend. Virus adsorption inhibitors interact with cationic regions of theviral coat protein, thereby preventing association with receptors of thepotential host cell. In contrast to the adsorption inhibitors, theinhibitors of virus cell fusion do not act as early as to preventbinding, but rather act at a later stage to prevent fusion with the hostcell to form a common membrane. Another way would be inhibition ofpenetration with liberation of the viral genome, as has been describedin the prior art, e.g. for Picorna viruses. Furthermore, it is possibleto block the transcription and protein biosynthesis of viruses. Methodsof inhibiting viral DNA polymerase have also been described in the priorart. The inhibition of viral DNA polymerase has been disclosed in theprior art particularly for herpes viruses. The DNA polymerase of herpesviruses assumes various functions. Among other things, it is responsiblefor the introduction of the viral genetic information into the host cellgenome, for RNA-dependent DNA synthesis, for DNA-dependent DNAsynthesis, and has additional functions. A large number of presentlyknown, successfully applied antiviral compounds are nucleoside-analogoussubstances which, however, are limited in their antiviral activity toherpes viruses in particular.

As the above-mentioned strategies are successful in herpes viruses, inparticular, and allow application to other viruses with less success insome cases, it has been necessary to develop different therapies foreach particular group of viruses. Thus, for example, vaccines producedby genetic engineering have been available for years for the treatmentof hepatitis B; however, they fail to be helpful in individuals alreadyinfected and exert significant influence on the above-mentioned chroniccourse of said disease. The nucleosides of the invention avoid theabove-specified drawbacks of the prior art.

Without intending to be limiting, the invention will be explained inmore detail with reference to the following examples.

EXAMPLES 1. Synthesis of 4-hydroxyaminopyrimidin-2(1H)-oneβ-L-Nucleosides from the Corresponding Uracil or Thymine Nucleosides 1.1Synthesis of1-(2-deoxy-β-L-ribofuranosyl)-4-hydroxyaminopyrimidin-2(1H)-one(β-L-N4-hydroxydeoxycytidine)

1-(2,3-Di-O-benzoyl-2-deoxy-β-L-ribofuranosyl)uracil (1.3 g, 2.98 mmol)was dissolved in triethylamine (1.8 ml, 12.9 mmol) and anhydrousacetonitrile (70 ml). The solution was cooled to 0° C. in an argonatmosphere and mixed with 2,4,6-triisopropylbenzenesulfonyl chloride(1.95 g, 6.3 mmol) and 4-dimethylaminopyridine (300 mg, 2 mmol). Thereaction mixture was left at room temperature overnight with stirring.Subsequently, hydroxylamine hydrochloride (450 mg, 6.47 mmol) was addedand the reaction solution was stirred at room temperature for 24 hours.Thereafter, water (50 ml) and chloroform (75 ml) were added. The organicphase was washed with saturated sodium chloride solution and dried oversodium sulfate. The residue obtained after removing the solvent invacuum was purified by means of column chromatography on silica gel,using chloroform/methanol (98/2, v/v) as eluent.1-(2,3-Di-O-benzoyl-β-L-ribofuranosyl)-4-hydroxyamino-pyrimidin-2(1H)-onewas isolated from the corresponding fractions as a white amorphous mass(1.7 g).

The above amount of substance was added to ammonia-saturated methanol(20 ml). The reaction solution was left for 24 hours at room temperatureand was subsequently concentrated to dryness in vacuum. The residue waspurified by means of column chromatography on silica gel, using achloroform/methanol (9/1, v/v) mobile phase.1-(2-Deoxy-β-L-ribofuranosyl)-4-hydroxyaminopyrimidin-2(1H)-one wasobtained from the corresponding fractions and crystallized frommethanol/ether (yield: 232 mg, 0.94 mmol, 31, 6%).

1.2 Synthesis of1-(2-Deoxy-β-L-ribofuranosyl)-4-hydroxyamino-5-methylpyrimidin-2(1H)-one(β-L-5-methyl-N4-hydroxydeoxycytidine)

According to the general synthetic method described above and startingfrom 1-(3,5-di-O-acetyl-2-deoxy-β-L-ribofuranosyl)thymine (500 mg, 1.53mmol), β-L-5-methyl-N4-hydroxydeoxycytidine was obtained (132 mg, 0.5mmol, 32%).

1.3 Synthesis of 1-(2-deoxy-β-L-ribofuranosyl)5-fluoro-4-hydroxyaminopyrimidin-2(1H)-one(β-L-5-fluoro-N4-hydroxydeoxycytidine)

β-L-5-Fluoro-2′-deoxyuridine was prepared according to establishedmethods for the synthesis of the corresponding D-derivative (Ozaki etal., Bull Chem Soc Japan 1977, 50: 2197-2198).

A stirred solution of1-(5-O-acetyl-2-deoxy-β-L-ribofuranosyl)-5-fluorouracil (288 mg, 1 mmol)in anhydrous acetonitrile (30 ml) under an argon atmosphere was cooledto 0° C. To this solution were successively added 2,4,6-triisopropylbenzenesulphonyl chloride (654 mg, 2.1 mmol) and 4-dimethylaminopyridine(132 mg, 1 mmol). The resulting mixture was stirred for 20 h at roomtemperature. Solid hydroxylamine hydrochloride (149 mg, 2.1 mmol) wasadded and the mixture was stirred for an additional 24 h. The mixturewas partitioned between water (25 ml) and chloroform (100 ml). Theorganic layer was washed with a saturated aqueous sodium chloridesolution (30 ml), dried over anhydrous sodium sulfate, filtered, and thesolvent was removed under reduced pressure. The resulting residue waspurified by column chromatography on silica gel eluting with a gradientof methanol (0-10%) in chloroform to afford1-(5-O-acetyl-2-deoxy-β-L-ribofuranosyl)-5-fluoro-4-hydroxyaminopyrimidin-2(1H)-oneas a white solid (138 mg, 0.45 mmol). A solution of this compound inmethanol saturated with ammonia at 0° C. was kept for 24 h at roomtemperature. After removing of the solvent under reduced pressure theresidue was purified by column chromatography on silica gel withchloroform/methanol (9/1, v/v) as eluent to afford1-(2-deoxy-β-L-ribofuranosyl)-5-fluoro-4-hydroxyaminopyrimidin-2(1H)-one(94 mg, 036 mmol) as a white solid.

1.4 Synthesis of1-(2,3-dideoxy-β-L-glycero-pentofuranosyl)-5-fluoro-4-hydroxyaminopyrimidin-2(1H)-one(β-L-2′, 3′-dideoxy-5-fluoro-N4-hydroxycytidine)

β-L-2′,3′-Didehydro-2,3′-dideoxy-5-fluorouridine was pre-pared accordingto established methods described for the synthesis of the correspondingD-derivative (Joshi et al., J Chem Soc Perkin Trans I 1992, 2537-2544).This compound was acetylated in the usual manner with acetanhydride inpyridine and purified by column chromatography. The isolated product wasactivated with 2,4,6-triisopropyl benzenesulphonyl chloride and4-dimethylaminopyridine, then reacted with solid hydroxylaminehydrochloride as described in example 1.3. The reaction product waspurified by column chromatography to afford the acetylated N4hydroxycytidine derivative.

A solution of1-(5-O-acetyl-2,3-dideoxy-β-L-glycero-pent-2-enofuranosyl)-5-fluoro-4-hydroxyaminopyrimidin-2(1H)-one(285 mg, 1 mmol) in dioxane was catalytically hydrogenolyzed asdescribed in example 1.6.

The product of that reaction was deacetylated by treatment with asolution of ammonia in methanol (saturated at 0° C.) for 24 h. Thesolvent was removed under reduced pressure. The residue was purified bycolumn chromatography on silica gel eluting with chloroform/methanol(95/5, v/v).1-(2,3-dideoxy-β-L-glycero-pentofuranosyl)-5-fluoro-4-hydroxyaminopyrimidin-2(1H)-onewas afforded as a white foam (67 mg, 0.27 mmol).

¹H-NMR (DMSO-d₆) δ 10.43, 9.99 (2H, s, NH-4, OH-4), 7.54 (1H, d, H-6),5.73 (1H, t, H-1′), 5.21 (1H, t, OH-5′), 4.23-4.18 (1H, m, H-4′),3.70-3.45 (2H, m, H-5′, H-5″), 2.17-2.04 (4H, m, H-3′, H-3″, H-2′,H-2′).

1.5 Synthesis of1-(2,3-dideoxy-β-L-glycero-pent-2-enofuranosyl)-4-hydroxyaminopyrimidin-2(1H)-one(β-L-2′,3′-didehydro-2′,3′-dideoxy-N4-hydroxycytidine)

β-L-2′,3′-Didehydro-2′,3′-dideoxyuridine was prepared according toestablished methods described for the synthesis of the correspondingD-derivative (Horwitz et al., J Org Chem 1966, 31:205-211).

1-(5-O-Acetyl-2,3-dideoxy-β-L-glycero-pent-2-enofuranosyl)-uracil (288mg, 1 mmol) was dissolved in dioxane (30 ml) cooled to 0° C. To thissolution were added successively under an argon atmosphere2,4,6-triisopropylbenzenesulphonyl chloride (654 mg, 2.1 mmol) and4-dimethylaminopyridine (132 mg, 1 mmol).

This solution was stirred for 24 h at room temperature. Hydroxylaminehydrochloride (149 mg, 2.1 mmol) was then added, and the mixture wasfurther stirred fox 1 day at room temperature. Water (25 ml) was added,and the product was extracted with chloroform (100 ml). The organiclayer was washed with a aqueous solution saturated with sodium chloride(30 ml), dried over anhydrous sodium sulfate, filtered, and concentratedunder reduced pressure.

The resulting residue was purified by column chromatography on silicagel eluting with a gradient of methanol (0-5%) in chloroform to give1-(5-O-acetyl-2,3-dideoxy-β-L-glycero-pent-2-enofuranosyl)-4-hydroxyaminopyrimidin-2(1H)-oneas a white foam. This compound was concentrated vacuo, the residue waspurified by column chromatography on silica gel eluting withchloroform/methanol (95/5, v/v) to afford1-(2,3-dideoxy-β-L-glycero-pent-2-enofuranosyl)-4-hydroxyaminopyrimidin-2(1H)-one(79 mg, 0.35 mmol).

¹H-NMR (DMSO-d₆) δ 10.96, 10.01 (2H, 2s, NH-4, OH-4), 7.64 (1H, s, H-6),6.58 (1H, d, H-1′), 6.37 (1H, dd, H-3′), 6.23 (1H, dd, H2′), 5.53 (1H,d, H-5), 5.21 (1H, t, OH-5′), 4.20-4.12 (1H, m, H-4′), 3.75-3.52 (2H, m,H-5′, H-5″).

1.6 Synthesis of1-(2,3-dideoxy-β-L-glycero-pentofuranosyl)-4-hydroxyaminopyrimidin-2(1H)-one(β-L-2′,3′-dideoxy-N4-hydroxycytidine)

β-L-2′,3′-Didehydro-2′,3′-dideoxyuridine was prepared according toestablished methods described for the synthesis of the correspondingD-derivative (Horwitz et al., J Org Chem 1966, 31:205-211).

This deoxyuridine derivative was acetylated with acetanhydride inpyridine. The reaction product was purified by column chromatography.The isolated derivative was activated with 2,4,6-triisopropylbenzenesulphonyl chloride and 4-dimethylaminopyridine in acetonitrile,then hydroxylamino chloride was added and the reaction mixture wasworked up as described in example 1.3. After evaporation of the solventthe acetylated hydroxycytidine derivative was purified by columnchromatography.

A solution of1-(5-O-acetyl-2,3-dideoxy-β-L-glycero-pent-2-enofuranosyl)-4-hydroxyaminopyrimidin-2(1H)-one(267 mg, 1 mmol) in dioxane containing 125 mg of 10% palladium-charcoalcatalyst was shaken with 1 atm. of hydrogen at room temperature. Thetheoretical uptake of hydrogen was realized in 0.5 h, the catalyst wasfiltered, and the filtrate was evaporated to dryness.

The residue was treated with methanol/ammonia (25 ml) overnight at roomtemperature. After removing the solvent the corresponding residue waspurified by column chromatography on silica gel with chloroform/methanol(9/1, v/v) as solvent to afford1-(2,3-dideoxy-β-L-glycero-pentofuranosyl)-4-hydroxyaminopyrimidin-2(1H)-one(105 mg, 0.46 mmol) as a solid.

¹H-NMR (DMSO-d₆) δ 10.41, 9.95 (2H, 2s, NH-4, OH-4), 7.54 (1H, d, H-6),5.73 (1H, d, H-5), 5.58 (1H, t, H-1′), 5.03 (1H, t, OH-5′), 4.94 (m, 1H,H-4′), 3.51 (m, 2H, H-5′, H-5), 2.31-2.56 (m, 4H, H-3′, H-3″, H-2′,H-2″).

1.7 Synthesis of1-(2,3-dideoxy-β-L-glycero-pent-2-eno-furanosyl)-5-fluoro-4-hydroxyaminopyrimidin-2(1H)-one(β-L-2′,3′-Didehydro-2′,3′-dideoxy-5-fluoro-N4-hydroxycytidine)

β-L-2′,3′-Didehydro-2′,3′-dideoxy-5-fluorouridine was prepared accordingestablished methods for synthesis of the corresponding D-derivative(Joshi et al., J Chem Soc Perkin Trans I 1992, 2537-2544).

In a similar manner as described under example 15 using1-(5-O-acetyl-2,3-dideoxy-β-L-glycero-pent-2-enofuranosyl)-5-fluorouracil(252 mg, 1 mmol) as starting material, the title compound1-(2,3-dideoxy-β-L-glycero-pent-2-enofuranosyl)-5-fluoro-4-hydroxyaminopyrimidin-2(1H)-onewas obtained (69 mg, 0.33 mmol).

1.8 Synthesis of1-(2,3-dideoxy-β-L-glycero-pent-2-enofuranosyl)-5-methyl-4-hydroxyaminopyrimidin-2(1H)-one

(β-L-2′,3′-didehydro-2′,3′-dideoxy-5-methyl-N4-hydroxycytidine)

2′,3′-Didehydro-2′,3′-deoxy-β-L-thymidine (5-L-thymidinene) was preparedaccording to established methods described for synthesis ofcorresponding D-derivative (Horwitz et al., J Org Chem 1966, 31:205-211). β-L-thymidinene was acetylated in the usual manner withacetanhydride in pyridine. 5′-O-acetyl-2′,3′-didehydro-2′,3′-deoxy-β-L-thymidine (266 mg, 1 mmol) was subjected to the samesequence of reaction steps as described in the example 1.5 to afford1-(2,3-dideoxy-5-L-glycero-pent-2-enofuranosyl)-5-methyl-4-hydroxy-aminopyrimidin-2(1H)-one(132 mg, 0.55 mmol).

¹H-NMR (DMSO-d₆) δ10.44, 10.02 (2H, s, NH-4, OH-4), 7.63 (1H, s, H-6),6.81 (1H, dd, H-1′), 6.42 (1H, m, H-3′), 5.95 (1H, m, H-2′), 5.02 (1H,brt, OH-5′), 4.78 (1H, m, H-4′), 3.62 (2H, m, H-5′, H-5″), 1.78 (3H, s,CH₃).

2. Synthesis of 4-hydroxyaminopyrimidin-2(1H)-one β-L-Nucleosides fromthe Corresponding Cytosine Nucleosides 2.1 Synthesis ofS-L-2′,3′-dideoxy-3′-thia-N4-hydroxycytidine

β-L-2′,3′-Dideoxy-3′-thiacytidine was synthesized as described (Beach etal., J Org Chem 1992, 57: 2217-2219). 500 mg (2.18 mmol) of it was mixedwith a 7 M hydroxylamine hydrochloride solution (25 ml). The reactionsolution was kept at room temperature for four days with stirring.Following removal of the solvent in vacuum, the resulting residue waspurified by means of column chromatography on silica gel, using theupper phase of the mixture ethyl acetate/isopropanol/water (4/1/2,v/v/v) as eluent.

The solvent of the corresponding fractions was removed in vacuum.β-L-2′,3′-dideoxy-3′-thia-N4-hydroxycytidine was obtained from themethanol solution of the residue (yield: 95 mg, 0.39 mmol, 17.9%).

2.2 Synthesis of1-(2,3-dideoxy-2-fluoro-β-L-glycero-pent-2-enofuranosyl)-4-hydroxyaminopyridin-2(1H)-one(β-L-2′,3′-didehydro-2,3′-dideoxy-2′-fluoro-N4-hydroxycytidine)

β-L-2′,3′-Didehydro-2′,3′-dideoxy-2′-fluorocytidine was synthesized asdescribed (Lee et al., J Med Chem 1999, 42:1320-1328).

400 mg (1.76 mmol) of this compound was dissolved in 10 ml of 5 Mhydroxylamine hydrochloride which had been adjusted to pH 6.0 withsodium hydroxide. The solution was stirred for 24 h at room temperature,and the solvent was removed under reduced pressure.

The residue was purified by column chromatography on silica gel withchloroform/methanol (9/1, v/v) as eluent to afford1-(2,3-dideoxy-2-fluoro-β-L-glycero-pent-2-enofuranosyl)-4-hydroxyaminopyridin-2(1H)one(83 mg, 0.34 mmol, yield 19.3%).

2.3 Synthesis ofβ-L-[2-(hydroxymethyl)-1,3-oxathiolan-4-yl]-5-fluoro-4-hydroxyaminopyridin-2(1H)-one(β-L-2′,3′-dideoxy-3′-thia-5-fluoro-N4-hydroxycytidine)

β-L-2,3′-Dideoxy-3′-thia-5-fluorocytidine was synthesized as described(Beach et al., J Org Chem 1992, 57: 2217-2219).

78 mg (0.31 mmol) of this compound was shaken for 24 h in 2 ml ofaqueous 5 M hydroxylamine hydrochloride (adjusted to pH 6.0).

The solvent was removed in vacuo, and the residue was purified by columnchromatography on silica gel eluting with chloroform/methanol (9/1,v/v). From the corresponding fractionsβ-L-[2-(hydroxymethyl)-1,3-oxa-thiolan-4-yl]-5-fluoro-4-hydroxyaminopyridin-2(1H)-onewas isolated as a foam (14 mg, 0.05 mmol, yield 16%).

2.4 Synthesis of1-(3-azido-2,3-dideoxy-β-L-ribofuranosyl)-4-hydroxyaminopyridin-2(1H)-one(β-L-3′-azido-2′,3′-dideoxy-N4-hydroxycytidine)

β-L-3-Azido-2′,3′dideoxycytidine was prepared according to establishedmethods described for the synthesis of the corresponding D-derivative.300 mg, (1.2 mmol) of this compound was dissolved in 10 ml aqueous 5 Mhydroxylamine hydrochloride (adjusted to pH 6.0) and treated accordingexample 2.2. The title compound was obtained as a white solid (103 mg,0.38 mmol, yield 31.6%).

2.5 Synthesis of1-(3-azido-2,3-dideoxy-β-L-ribofuranosyl)-5-fluoro-4-hydroxyaminopyridin-2(1H)-one(β-L-3′-azido-2′,3′-dideoxy-5-fluoro-N4-hydroxycytidine)

β-L-3′-Azido-2′,3′dideoxy-5-fluorocytidine was prepared according toestablished methods described for the synthesis of the correspondingD-derivative (Sandström et al., Drugs 1986, 31: 462-467).

500 mg (1.85 mmol) of this compound were treated as described in example2.2. The title compoundβ-L-3′-azido-2′,3′-dideoxy-5-fluoro-N4-hydroxycytidine (121 mg, 0.42mmol, yield 22.7%) was obtained.

2.6 Synthesis of1-(3-azido-2,3-dideoxy-β-L-ribofuranosyl)-5-methyl-4-hydroxyaminopyridin-2(1H)-one(β-L-3-azido-2′,3′-dideoxy-5-methyl-N4-hydroxycytidine)

β-L-3′-Azido-2′, 3′dideoxy-5-methylcytidine was prepared according toestablished methods described for the synthesis of the correspondingD-derivative (Lin et al., J Med Chem 1983, 26: 544-551).

450 mg, (1.69 mmol) of this compound were treated as described inexample 2.2. The title compoundβ-L-3′-azido-2′,3′-dideoxy-5-methyl-N4-hydroxycytidine (143 mg, 0.5mmol, yield 29.5%) was obtained.

2.7 Synthesis of1-(2,3-dideoxy-3-fluoro-β-L-ribofuranosyl)-4-hydroxyaminopyridin-2(1H)-one(β-L-2′,3′-dideoxy-3′-fluoro-N4-hydroxycytidine)

β-L-2′,3′-Dideoxy-3′-fluorocytidine was synthesized as described (vonJanta-Lipinski et al. J Med Chem 1998, 12: 2040-2046.)

This compound (350 mg, 1.52 mmol) gave according to the synthetic methoddescribed in example 2.2,1-(2,3-dideoxy-3-fluoro-β-L-ribofuranosyl)-4-hydroxyaminopyridin-2(1H)-one(137 mg, 0.56 mmol, yield 36.8%) as a solid.

¹H-NMR (DMSO-d₆) δ 10.48, 10.06 (2H, s, NH-4, OH-4), 7.76 (1H, d, H-6),6.25 (1H, m, H-1′), 5.47 (1H, d, H-5), 5.25 (1H, dd, H-3′, J_(F-3)=53.6Hz), 5.11 (1H, t, OH-5′), 4.13 (1H, dt, H-4′, J_(F-4)=27 Hz), 3.52-3.64(2H, m, H-5′, H-5″), 2.38-2.45 (2H, m, H-2′, H-2″).

3. Inhibition of HBV-Replication by the Compounds of Invention in HepG22.2.15 Cells

The antiviral efficacy of the compounds of the invention wasinvestigated on HepG2 2.2.15 cells, a human hepatoblastoma cell linewhich has the replication-competent HBV genome stably integrated thereinand produces infectious progeny viruses in a productive manner (Sells etal., Proc Natl Acad Sci USA 1987, 84: 1005-1009).

The above cells were cultured under standardized conditions as specifiedby Korba and Gerin, and the amount of extracellular viral DNA wasdetermined (Korba et al., Antiviral Res 1992, 19: 55-70).

Following passaging, the HepG2 2.2.15 cells were seeded at a density ofabout 60% in 12-well plates and cultured to confluence in 10% FBSDulbecco MEM. Thereafter, the medium was changed to 2% FBS, and thecells were cultured for another 24 hours.

After another change of medium, the cells were treated with varyingconcentrations of compounds according to the invention. Every 24 hoursthe compounds were re-added together with the medium. On the 6^(th) dayof treatment, the cell supernatants were centrifuged off and stored at−20° C. until analysis of the HBV DNA was effected.

Following treatment of the culture supernatants with proteinase K, theextracellular viral DNA was amplified by means of PCR using thefollowing primers (forward: 5′-CTC CAG TTC AGG AAC AGT AAA CCC-3′;reverse: 5′-TTG TGA GCT CAG AAA GGC CTT GTA AGT TGG CG-31. The PCRproducts were separated on 1% agarose, stained with ethidium bromide andquantified using a Fluor-S™ Multimager (Biorad).

For calibration of the PCR reaction, serial dilutions of the pUC19 HBVand pTHBV plasmids with known genome equivalents (GE) were used,resulting in a lower detection limit of about 10³ GE and a linearitybetween 10³ and 10⁵ GE. Table 1 shows the concentrations of compounds ofthe invention required for 50% reduction of extracellular HBV DNA (ED₅₀)after 6 days incubation of the HepG2 2.2.15 cells.

Between the new compoundsβ-L-2′,3′-didehydro-2′,3′-dideoxy-N4-hydroxycytidine (L-HyddeC),5-L-2′,3′-didehydro-2′,3′-dideoxy-5-fluoro-N4-hydroxycytidine(L-HyddeFC) andβ-2′,3′-didehydro-2′,3′-dideoxy-2′-fluoro-N4-hydroxycytidine (L-HyFddeC)were the most effective nucleoside with EC₅₀-values of <0.1 μM.

A second group of compounds includingβ-L-2′-3′-dideoxy-3′-thia-N4-hydroxycytidine (Hy3TC),β-L-2′-3′-dideoxy-3′-thia-5-fluoro-N4-hydroxycytidine (HyFTC),β-L-2′,3′-dideoxy-N4-hydroxycytidine (L-HyddC), andβ-L-2′,3′-dideoxy-5-fluoro-N4-hydroxycytidine (L-HyddFC) gaveEC₅₀-values between 0.3 and 0.65 μM.

A third group of compounds of the invention with EC₅₀-values between 3and 50 μM includes β-L-N4-hydroxydeoxycytidine (L-HyCdR),β-L-5-fluoro-N4-hydroxydeoxycytidine (L-HyFCdR),β-L-5-methyl-N4-hydroxydeoxycytidine (L-HyMetCdR),β-L-3′-fluoro-2′,3′-dideoxy-N4-hydroxycytidine (L-FHyCdR), andβ-L-3′-azido-2′, 3′-dideoxy-N4-hydroxycytidine (L-N₃HyCdR).

It can be argued that the N-4-hydroxy-group of the β-L-cytidinederivatives could be metabolized inside of cells to the correspondingNH₂-group. Such a reaction could suggest a prodrug function of thepresented analogues. In this case Hy3TC as the prodrug of 3TC shouldalso display a high efficiency against HIV because 3TC inhibits theHIV-replication at a EC₅₀ of 0.002 μM (Schinazi et al., AntimicrobAgents Chemother 1992, 38: 2423-2431).

However, we found that Hy3TC is inactive against HIV replication(EC₅₀>>25 μM) ruling out the possibility that the metabolic conversionof the NHOH-group to the NH₂-group could be the reason for its anti-HBVactivity.

TABLE 1 Inhibition of HBV-replication in HepG2 2.2.15 cells by β-L-hydroxycytosine nucleosides compared to 3TC(lamivudine), β-L-dideoxycytidine(L-ddC), β-L-thymidine(L-TdR), β-L-5-fluorode-oxycytidine(L-FCdR). The concentrations required for 50% reduction ofHBV DNA in the medium of the cells are given(EC₅₀; μM). Compound EC₅₀;μM 3TC (lamivudine) 0.1 L-ddC 0.25 L-TdR 0.3 L-FCdR 1.2 L-HyCdR 3.0L-HyFCdR 4.5 L-HyMetCdR 7.8 L-FHyCdR 25 L-N₃HyCdR 50 Hy3TC 0.5 HyFTC 0.3L-HyddC 0.65 L-HyddFC 0.35 L-HyddeC 0.05 L-HyddeFC <0.1 L-HyFddeC <0.1Abbreviations: 3TC (lamivudine) = 2,3′-dideoxy-3′-thiacytidine; L-HyCdR= β-L-N4-hydroxydeoxycytidine; L-HyFCdR =β-L-5-fluoro-N4-hydroxydeoxycytidine; L-HyMetCdR =β-L-5-methyl-N4-hydroxydeoxycytidine; L-FHyCdR =β-L-3′-fluoro-2′,3′-dideoxy-N4-hydroxycytidine; L-N₃HyCdR =β-L-3′-azido-2′,3′di-deoxy-N4-hydroxycytidine; Hy3TC =β-L-2′-3′-dideoxy-3′-thia-N4-hydroxycytidine; HyFTC =β-L-2′-3′-dideoxy-3′-thia-5-fluoro-N4-hydroxycytidine; L-HyddC =β-L-2′,3′-dideoxy-N4-hydroxycytidine; L-HyddFC =β-L-2′,3′-dideoxy-5-fluoro-N4-hydroxycytidine; L-HyddeC =β-L-2′,3′-didehydro-2′,3′-dideoxy-N4-hydroxycytidine; L-HyddeFC =β-L-2′,3′-didehydro-2′,3′-dideoxy-5-fluo-ro-N4-hydroxycytidine;L-HyFddeC =β-L-2′,3′-didehydro-2′,3′-dideoxy-2′-fluoro-N4-hydroxycytidine.

4. Inhibition of HBV DNA Polymerase by β-L-N4-hydroxycytosine NucleosideTriphosphates

Synthesis and purification of the triphosphates ofβ-L-N4-hydroxycytosine nucleosides were performed according towell-known methods (Yoshikawa et al., Tetrahedron Lett 1967, 50:5065-5068; Hoard et Ott, J Am Chem Soc 1965, 87: 1785-1788).

To determine the endogenous HBV DNA polymerase activity, about 100 ml ofserum from patients with untreated hepatitis B virus infections fromCharité, Berlin, (>10⁷ HBV particles/ml), was centrifuged at 3000 rpm.Virus particles of the cleared serum were sedimented in a Beckman SW28rotor at 25,000 rpm, 60 min. The virus pellet was suspended in 7 ml ofTKM buffer (50 mM Tris-HCl, pH 7.5, 50 mM KCl, 5 mM MgCl₂), Layered overa step gradient of 10 ml each of 0.3 M, 0.6 M, 0.9 M saccharose in TKMbuffer and centrifuged at 25,000 rpm for 20 hours.

The purified virus pellet was suspended in 250 μl TKM buffer, lysed byultrasound, divided in aliquots and frozen at −80° C. (Davies et al.,Antiviral Res 1996, 30: 133-145).

The HBV DNA polymerase assay contained in 30 μl about 2-4×10⁸ purifiedvirus particles (lysed beforehand additionally in 6% β-mercaptoethanol,10% Igepal for 15 min at room temperature), 42 mM Tris-HCl, (pH 7.5), 34mM MgCl₂, 340 mM KCl, 22 mM β-mercaptoethanol, 0.4% Igepal, 70 μM TTP,dATP, dGTP and 1 μCi ³H-dCTP (=0.7 μM dCTP) (Matthes et al., AntimicrobAgents & Chemother 1991, 35: 1254-1257) and varying concentrations ofβ-L-N4-hydroxycytosine nucleoside triphosphates as inhibitors.

Following a one-hour incubation at 37° C., 20 μl of the assay volume wasplaced on paper filter, washed 5 times with 5% trichloroacetic acid and0.1% Na pyrophosphate, and the ³H-dCMP incorporated in the HBV DNA wassubsequently measured in a Liquid Scintillation Counter.

Using the concentration-dependent inhibition curves of HBV DNAsynthesis, the concentration of 1-L-N4-hydroxycytosine nucleosidetriphosphates resulting in 50% inhibition of the HBV DNA polymeraseactivity was determined.

Table 2 demonstrates that the HBV DNA polymerase is inhibited stronglyby the triphosphates of L-Hy3TC, L-HyddC and L-HyddeC (IC₅₀ between 0.15and 0.65 μM) pointing out that the 4-NHOH-group of the cytosinenucleoside triphosphates is effective at the target and does not requirea previous metabolization to the NH₂-group.

TABLE 2 Inhibition of HBV DNA polymerase by triphosphates of β-L-hydroxycytidine nucleoside analogues in comparison to 3TC-tri-phosphate(IC₅₀). Triphosphate of IC₅₀; μM 3TC (lamivudine) 0.30 L-HyCdR6.0 L-HyMetCdR 4.0 L-Hy3TC 0.65 L-HyddC 0.55 L-HyddeC 0.28

5. Cytotoxicity of D-L-N4-hydroxycytosine Nucleosides

To this end, established cells of a human myeloid leukemia (HL-60) inRPMI medium, and the above-mentioned HepG2 cells in Dulbecco MEM,respectively, were incubated for two days using varying concentrationsof compounds, and the proliferation rate of the cells was subsequentlydetermined. The data were used to determine the concentration ofcompounds resulting in 50% inhibition of proliferation (CD₅₀). Table 3shows that the new compounds display no antiproliferative activity onHepG2- and HL-60 cells.

Remarkably, also L-HyddC, L-HyddeFC and L-HyFddeC have lost theantiproliferative activity which was described for the correspondingcytosine analogues containing the q-NH₂-group instead of the4-NHOH-group (IC₅₀ for L-ddC=70 μM, Lin et al., J Med Chem 1994, 37:798-803; IC₅₀ for ddeFC=7 μM, Lin et al., J Med Chem 1996, 39:1757-1759; IC₅₀ for L-FddeC=100 μM, Lee et al., J Med Chem 1999, 42:1320-1328).

Thus these data further support our suggestion that the NH₂-group couldnot be formed inside of cells from our β-L-N4-hydroxycytosine nucleosideanalogues.

TABLE 3 Cytotoxicity of β-L-N4-hydroxycytosine-nucleosides againstHepG2-and HL-60 cells in comparison to 3TC(lamivudine). Concentrationsproducing 50% inhibition of cell proliferation were given (CD₅₀). CD₅₀;μM Compound HepG2-cells HL-60 cells 3TC (lamivudine) 1900 2000 L-HyCdR545 460 L-HyFCdR 920 1370 L-HyMetCdR 490 600 L-FhyCdR 820 4400 L-N₃HyCdR1160 2000 Hy3TC 1230 1450 HyFTC 975 1100 L-HyddC 2250 1600 L-HyddFC 15801860 L-HyddeC >5000 >3500 L-HyFddeC 1370 1130 Abbreviations: 3TC(lamivudine) = 2,3′-dideoxy-3′-thiacytidine; L-HyCdR =β-L-N4-hydroxydeoxycytidine; L-HyFCdR =β-L-5-fluoro-N4-hydroxydeoxycytidine; L-HyMetCdR =β-L-5-methyl-N4-hydroxydeoxycytidine; L-FHyCdR =β-L-3′-fluoro-2′,3′-dideoxy-N4-hydroxycytidine; L-N₃HyCdR =β-L-3′-azido-2′,3′-di-deoxy-N4-hydroxycytidine; Hy3TC =β-L-2′-3′-dideoxy-3′-thia-N4-hydroxycytidine; HyFTC =β-L-2′-3′-dideoxy-3′-thia-5-fluoro-N4-hydroxycytidine; L-HyddC =β-L-2′,3′-dideoxy-N4-hydroxycytidine; L-HyddFC =β-L-2′,3′-dideoxy-5-fluoro-N4-hydroxycytidine; L-HyddeC =β-L-2′,3′-didehydro-2′,3′-dideoxy-N4-hydroxycytidine; L-HyddeFC =β-L-2′,3′-didehydro-2′,3′-dideoxy-5-fluoro-N4-hydroxycytidine; L-HyFddeC= β-L-2′,3′-didehydro-2′,3′-dideoxy-2′-fluoro-N4-hydroxycytidine.

The state of the art may disclose more common empirical formulae, whichdo not however describe the special, chosen chemical compounds of thedoctrine according to the application. Those special, precise compoundsof the invention had not yet been made accessible in the form of commonterms and conceptions, since it was not possible to generate exactly thecompounds of the invention only by conducting routine experiments; thosecompounds show surprising, unobvious characteristics, for example thefact that hitherto all efforts of experts in this matter were in vain, adifferent approach to the development of scientific technology, theachievement forwards the development, misconceptions about the solutionof the according problem (prejudice), technical progress (such as:improvement, increased performance, price-reduction, saving of time,material, work steps, costs or resources that are difficult to obtain,improved reliability, remedy of defects, improved quality, increasedefficiency, augmentation of technical or medical possibilities,provision of another product, spare product, alternatives, enrichment ofthe pharmaceutical fund), a special choice (since a certain possibility,the result of which was unforeseeable, was chosen among a great numberof possibilities).

The precise, claimed chemical compounds of the application have not yetbeen disclosed in greater fields that are comprised by a common formula.The precisely chosen compounds of the invention are not arbitrarilychosen specimen, but it is rather the selective choice that leads toproducts with the above-mentioned surprising characteristics.

1. New β-L-N4-hydroxycytosine deoxynucleotides of general formula I forthe treatment and prophylaxis of HBV and HIV infections

wherein: R═H, halogen (F, Cl, Br, I), C₁-C₃ alkyl, and

wherein R₁═H, F; R₂═H, F, OH, N₃; and R₃═OH, O-acetyl, O-palmitoyl,alkoxycarbonyl, carbamate, phosphonate, monophosphate,bis(S-acyl-2-thioethyl) phosphate, diphosphate or triphosphate.
 2. Theβ-L-nucleosides according to claim 1, wherein R═H, F, Cl, Br, I, or CH₃.3. The β-L-nucleosides according to claim 1, wherein R═H, F or CH₃, andR₁═H or F, preferably H, R₂═H, F, OH or N₃, and R₃═OH.
 4. Aβ-L-nucleoside according to claim 1, said β-L-nucleoside beingβ-L-N4-hydroxydeoxycytidine, β-L-5-methyl-N4-hydroxydeoxycytidine,β-L-5-fluoro-N4-hydroxydeoxycytidine,β-L-2′,3′-dideoxy-N4-hydroxycytidine,β-L-2′,3′-dideoxy-5-fluoro-N4-hydroxycytidine,β-L-2′,3′-didehydro-2′,3′-dideoxy-N4-hydroxycytidine,β-L-2′,3′-didehydro-2′,3′-dideoxy-5-fluoro-N4-hydroxycytidine,β-L-2′,3′-didehydro-2′,3′-dideoxy-5-methyl-N4-hydroxycytidine,β-L-2′,3′-didehydro-2′,3′-dideoxy-2′-fluoro-N4-hydroxycytidine,β-L-2′,3′-dideoxy-3′-thia-N4-hydroxycytidine,β-L-2′,3′-dideoxy-3′-thia-5-fluoro-N4-hydroxycytidine,β-L-3′-azido-2′,3′-dideoxy-N4-hydroxycytidine,β-L-3′-azido-2′,3′-dideoxy-5-fluoro-N4-hydroxycytidine,β-L-3′-azido-2′,3′-dideoxy-5-methyl-N4-hydroxycytidine, andβ-L-3′-fluoro-2′,3′-dideoxy-N4-hydroxycytidine.
 5. The β-L-nucleosideaccording to claim 1, selected from the group consisting of a salt, aphosphonate, a monophosphate, bis(S-acyl-2-thioethyl) phosphate,diphosphate, triphosphate, an other ester and a salt of such ester. 6.The β-L-nucleoside according to claim 1 for producing a drug for thetreatment and prophylaxis of HBV and HIV infections. 7.Immunostimulatory nucleic acids or oligonucleotides for treatment ofcancer, HBV- and HIV-infections, asthma and allergic diseases containinga central de-oxycytidyl-deoxyguanosine dinucleotide (CpG) in which thedeoxycytidine is replaced by β-L-N4-hydroxydeoxycytidine,β-L-5-methyl-N4-hydroxydeoxycytidine, orβ-L-5-fluoro-N4-hydroxydeoxycytidine.
 8. A pharmaceutical agentcomprising a β-L-nucleoside or a derivative according to claim 1 and/ora nucleic acid containing a central de-oxycytidyl-deoxyguanosinedinucleotide (CpG) in which the deoxycytidine is replaced byβ-L-N4-hydroxydeoxycytidine, β-L-5-methyl-N4-hydroxydeoxycytidine, orβ-L-5-fluoro-N4-hydroxydeoxycytidine, optionally together withconventional auxiliaries, preferably carriers, adjuvants and/orvehicles.
 9. The pharmaceutical agent according to claim 8, furthercomprising one or more additional agents from the group of antiviral,fungicidal or antibacterial agents, anti-cancer agents and/orimmunostimulators or immunomodulators.
 10. The pharmaceutical agentaccording to claim 9, wherein the antiviral agents are proteaseinhibitors and/or reverse transcriptase inhibitors and/or inhibitors ofHBV DNA polymerase, the immunostimulators bropirimine, anti-humanalpha-interferon antibodies, IL-2, GM-CSF, interferons, diethyldithiocarbamate, tumor necrosis factors, naltrexone, tuscarasol and/orrEPO.
 11. The pharmaceutical agent according to claim 8, comprising oneor more additional anti-HBV-effective agents from the group comprisingPMEA (adefovir-dipivoxil), famciclovir, penciclovir,diaminopurine-dioxolane (DAPD), clevudine (L-FMAU), telbivudine(L-Thymidine) entecavir, interferon or thymosin al and/or inhibitors ofnucleocapsid formation, particularly heteroarylpyrimidines.
 12. Thepharmaceutical agent according to claim 8, wherein the agents arepegylated.
 13. The pharmaceutical agent according to claim 8 furthercomprising one or more additional agents capable of eliminating thefunction of cellular proteins essential to HBV growth.
 14. Thepharmaceutical agent according to claim 8, wherein said agent iseffective against hepatitis B viruses resistant to lamivudine or othercytosine nucleosides such as emtricitabine (L-FTC), L-ddC, L-ddeC, L-dCand/or elvucitabine (L-Fd4C).
 15. The pharmaceutical agent according toclaim 8, wherein said agent prevents cancer.
 16. The pharmaceuticalagent according to claim 8, wherein said agent prevents formation ofliver carcinoma resulting from chronic hepatitis triggered by HBV. 17.The pharmaceutical agent according to claim 8, wherein the carriers areselected from the group comprising fillers, diluents, binders,humectants, disintegrants, dissolution retarders, absorption enhancers,wetting agents, adsorbents and/or lubricants.
 18. A method forprophylaxis or therapy of a viral, bacterial, fungicidal and/orparasitic infection, or of cancer comprising: administering to a personin need of such prophylaxis or therapy the β-L-nucleosides of claim 1 ina viral, bacterial, fungicidal and/or parasitic infection, or cancerprophylaxis or therapy effective amount.
 19. The method of claim 18,wherein the viral infection is associated with hepatitis virus, HIV,bovine immunodeficiency virus, caprine arthritis-encephalitis virus,equine infectious anemia virus, ovine Maedi-Visna virus, Visna-Lentivirus, avian leukosis virus, human T cell leukemia virus, and/or felineimmunodeficiency virus.
 20. The method of claim 19, wherein thehepatitis virus is a hepatitis B or hepatitis D virus.
 21. The method ofclaim 19, wherein the HIV is HIV-0, HIV-1 and/or HIV-2.
 22. Theβ-L-nucleoside according to claim 1, wherein said β-L-nucleoside is aprodrug, a feed additive and/or a drinking water additive.
 23. Themethod of claim 1, wherein the said β-L-nucleoside is in form of a gel,poudrage, powder, tablet, sustained-release tablet, premix, emulsion,brew-up formulation, drops, concentrate, granulate, syrup, pellet,bolus, capsule, aerosol, spray and/or inhalant.
 24. The β-L-nucleosideaccording to claim 1, wherein said β-L-nucleoside is present in apreparation at a concentration of from 0.1 to 99.5, preferably from 0.5to 95, more preferably from 20 to 80 wt.-%.
 25. The method of claim 18,wherein said β-L-nucleoside is administered orally, rectally,subcutaneously, intravenously, intramuscularly, intraperitoneally and/ortopically mute.
 26. The method of claim 18, wherein said β-L-nucleosideis administered in overall amounts of from 0.05 to 500 mg/kg, preferablyfrom 1 to 100 mg/kg body weight per 24 hours.
 27. The method of claim18, wherein the β-L-nucleoside and/or a nucleic acid containing acentral de-oxycytidyl-deoxyguanosine dinucleotide (CpG) in which thedeoxycytidine is replaced by β-L-N4-hydroxydeoxycytidine,β-L-5-methyl-N4-hydroxydeoxycytidine, orβ-L-5-fluoro-N4-hydroxydeoxycytidine are employed administered in asingle administration of from 1 to 80, preferably from 3 to 30 mg/kgbody weight.
 28. The method of claim 18, wherein said β-L-nucleoside isadministered distributed over 2 to 10, preferably 3 to 5 dailyapplications.
 29. The method of claim 25, wherein 1 to 2 tablets areadministered in each oral application.
 30. The method of claim 18,wherein said β-L-nucleoside is used in combination with at least oneother well-known pharmaceutical agent.
 31. The method of claim 30,wherein said β-L-nucleoside enhances the therapeutic effect of saidother pharmaceutical agents in a non-additive, additive or synergisticfashion, increase the therapeutic index and/or reduce the risk oftoxicity inherent in the respective compound.
 32. The method of claim30, wherein said β-L-nucleoside is administered together with said otherwell-known pharmaceutical agents at a ratio of about 0.005 to
 1. 33. Themethod of claim 30, wherein said at least one β-L-nucleoside is used incombination with 3-deazauridine.
 34. (canceled)
 35. (canceled)
 36. A kitcomprising (a) the β-L-nucleoside according to claim 1 and/or (b) anucleic acid containing a central de-oxycytidyl-deoxyguanosinedinucleotide (CpG) in which the deoxycytidine is replaced byβ-L-N4-hydroxydeoxycytidine, β-L-5-methyl-N4-hydroxydeoxycytidine, orβ-L-5-fluoro-N4-hydroxydeoxycytidine and/or the pharmaceutical agentcomprising (a) and/or (b) optionally comprising a β-L-nucleoside or aderivative according to claim 1 and/or a nucleic acid containing acentral de-oxycytidyl-deoxyguanosine dinucleotide (CpG) in which thedeoxycytidine is replaced by β-L-N4-hydroxydeoxycytidine,β-L-5-methyl-N4-hydroxydeoxycytidine, orβ-L-5-fluoro-N4-hydroxydeoxycytidine, optionally together withconventional auxiliaries, Preferably carriers, adjuvants and/orvehicles, optionally together as well as with information for combiningthe contents of the kit.
 37. Use of the kit according to claim 36prophylaxis or therapy of viral diseases.
 38. A pharmaceuticalcombination preparation comprising at least one β-L-nucleoside accordingto claim 1 and 3-deazauridine for the treatment and/or prophylaxis ofHBV and HIV infections.